Methods of using αGal oligosaccharides as immune system targeting agents

ABSTRACT

The invention relates to methods for attenuating xenograft rejection in humans and old world monkeys, using oligosaccharides containing a Galα1-3Gal motif, to neutralize or remove anti-αGal antibodies. The invention additionally relates to methods for site directed activation of the complement cascade or host leukocytes using oligosaccharides containing a Galα1-3Gal motif to target anti-αGal antibodies. The invention further relates to pharmaceutical compositions that may be used in the practice of the invention. Such compositions contain, as the active ingredient, an oligosaccharide containing a Galα1-3Gal motif effective in binding anti-αGal antibodies in vivo or ex vivo.

1. INTRODUCTION

The invention relates to methods for attenuating xenograft rejection inhumans and old world monkeys, using oligosaccharides containing aGalα1-3Gal motif, to neutralize or remove anti-αGal antibodies. Theinvention additionally relates to methods for site directed activationof the complement cascade using oligosaccharides containing a Galα1-3Galmotif to target anti-αGal antibodies. The invention further relates topharmaceutical compositions that may be used in the practice of theinvention. Such compositions contain, as the active ingredient, anoligosaccharide containing a Galα1-3Gal motif effective in bindinganti-αGal antibodies in vivo or ex vivo.

2. BACKGROUND OF THE INVENTION

2.1. XENOGRAFT REJECTION

Advances in organ transplantation surgery and the development ofeffective immunosuppressive drug regimens has made organ transplantationa nearly routine procedure. The shortage of human donor organs is theprincipal obstacle in the transplantation field. Only a fraction oftransplantation candidates receive grafts, and many patients are noteven listed as candidates owing to this shortage. In addition, a numberof diseases (e.g., diabetes) do not include transplantation as a viableoption at this time. However, this perspective could change iftransplantation options were more permissive. Accordingly, muchattention has recently been placed on alternative animal organ donorsources. Higher primates are immunologically most suitable and have beenused as organ donors in a few cases, but are difficult and uneconomicalto breed, may impose a high risk of viral transmission and theirwidescale use in clinical transplantation is likely to raise ethicalobjections. Consequently, focus has been placed upon use of the pig asan organ donor. Swine constitute an attractive source of organ donorsfor clinical transplantation because they are plentiful, can be easilybred in captivity, have anatomical and physiologic compatibility withhumans and are amenable to genetic manipulation (Cooper et al., 1991, inXenotransplantation: the transplantation of organs and tissues betweenspecies, 481-500 (Springer, Berlin); Tumbleson, M. E. (ed.) 1985, Swinein biomedical Research, Volume 3 (Plenum, N.Y.); Stanton et al. (eds.),1986 Swine in Cardiovascular Research, Vol. I-III (CRC Press, Florida)).

Transplantation between individuals of the same species or betweenclosely related species is called concordant, and between more distantspecies, discordant. The management of concordant graft rejection is nowpossible with immunosuppressive therapy. In contrast, discordanttransplantation, such as that between pig and human or old world monkey,is characterized by hyperacute rejection ("HAR"), an extremely rapidimmunological attack by preformed host antibodies which recognizemolecular structures expressed on the endothelial cell surface ofvascularized grafts (Starzl et al., 1993, Lancet 341:65; Auchincloss, H.1988, Transplantation 46:1; Tuso et al., 1993, Transplantation 56:651;Inverardi et al., 1994, Immunol Rev. 141:71-93). Vascularized graftsperformed between discordant species undergo hyperacute rejection withinminutes of implant and can lead to graft destruction withinapproximately 5-20 minutes in the case of a swine to old world monkeytransplantation. The mechanisms that mediate hyperacute rejection arenot susceptible to conventional immunosuppressive therapy (Auchincloss,H. 1988, Transplantation 46:1). Recent studies have suggested that ifHAR is weathered by the transplanted organ, the transplanted organ"accommodates" to the host, and its long-term survival becomesmanageable by more conventional immunosuppressive drugs (Platt, J.,1994, Immunol. Rev. 141:127-149; Bach et al., 1991, Transpl. Proc.23(1):205-207). There is therefore a great need for developinginnovative methods and compositions capable of achieving clinicallysignificant prolongation of xenograft function and survival byovercoming hyperacute rejection (Platt et al., 1990, Immunol. Today11:450).

In swine to old world monkey combinations, the recognition and bindingof antigens expressed on the endothelium of the donor organ by preformedxenoreactive IgM antibodies of recipient origin is considered the majorimmediate mediator of graft endothelial cell injury throughcomplement-dependent hyperacute rejection (Platt et al., 1991,Transplantation 52:214; Dalmasso et al, 1992, Immunopharmacology24:149). This role of xenoreactive antibodies in the immediaterecognition of a xenogeneic organ is suggested by observations that:perfusion of xenogeneic organs results in the selective depletion ofxenoreactive natural antibodies from the blood (Perper et al., 1966,Transplantation 4:337-388; Platt et al., 1990, Transplantation49:1000-1001; Giles et al., 1970, Transplant Proc. 2:522-537; Cooper etal., 1988, J. Heart Transplant 7:238-246; Fischel et al., 1992, J. HeartLung Transplant 11:965-974; Holzknecht et al., 1995, J. Immunol.154:4565-4575), depletion of xenoreactive antibodies through perfusionof xenogeneic organs delays hyperacute rejection of a xenograft evenwhen the complement system remains intact (Dalmasso et al., 1992, Am. J.Pathol. 140:1157-1166), hyperacute rejection does not occur when swinehearts are transplanted into newborn old world monkeys which have anintact complement system but very low levels of natural antibodies(Kaplan et al., 1994, Transplantation 59:1-6), infusion of antidonorantibodies may initiate the rejection of a xenogeneic organ graft(Perper et al., 1967, Transplantation 5:514-533; Chavez-Peon et al.,1971, Transplant Proc. 3:573-576) and specific inhibition of the bindingof natural antibodies delays the onset of hyperacute rejection (Gamblezet al., 1992, Transplantation 54:577-583; Ye et al., 1994Transplantation 58:330-337).

The histo-blood group A and B epitopes, against which anti-A and anti-Bantibodies are directed, are structurally defined trisaccharides (Lloydet al., 1968, Biochemistry 7:2976; Watkins, W. M., 1974, Biochem. Soc.Symp. 40:125; Watkins, W. M., 1980, Biochemistry and genetics of theABO, Lewis and P blood group systems, In: Advances in Human Genetics,Harris and Hirschhorn (eds), Vol. 10, Plenum, New York, p. 1). Baboons"hyperimmunized" to the incompatible donor group through intravenousinjection of a composition containing the incompatible donortrisaccharide reject heterotopic allografted ABO-incompatible donorhearts through hyperacute antibody-mediated vascular rejection within amean of 19 minutes. Continuous intravenous infusion of the incompatibleA or B donor group trisaccharide and/or ex vivo depletion with thisimmobilized trisaccharide, beginning immediately pre-transplantation andcontinued post-transplantation for several days, has been observed toprolong allograft survival to a mean of 8 days (Cooper et al., 1993,Transplantation, 56:769-777). While these results have led tospeculation that the ABO system serves as a model for HAR of xenografts,unlike the group A and B epitopes, the epitope(s) bound by anti-animalantibodies that are determinative of xenograft rejection, have not beenstructurally characterized thoroughly and effective anti-animal antibodyblocking substances have not been described.

Xenoreactive natural antibodies have been shown to play a major role ininitiating HAR in the case of old world monkey rejection of a swinexenograft, since their depletion appears to prevent complementactivation and abrogates HAR, potentially allowing prolongation ofxenograft survival for variable periods (Lu et al., 1994, FASEB, J.8:1122-1130; Platt et al., 1990, Transplantation 50:817-822). On theother hand, rejection of vascularized discordant xenogeneic organsinevitably takes place after these treatments, suggesting that othermechanisms must be involved in the recognition of the grafts. Forexample, an induced antibody response may take place, due tosensitization of the recipient (Valvidia et al., 1990, Transplantation50:132; Monden et al., 1989, Surgery 105:535; Bouwman et al., 1989,Transplant Proc. 21:551; Bouwman et al., Transplant Proc. 21:540; Sachset al., 1971, J. Immunol. 107:481). Additionally, the alternativepathway for complement activation, which can act in the absence ofantibodies, has also been implicated in xenograft rejection and may becapable of at least partially substituting for the directantibody-dependent pathway (Zhao et al., 1994, Transplantation 57:245;Forty et al., 1993, J. Heart Lung Transpl. 12:283; Wang et al., 1992,Histochem. T. 24:102; Miygawa et al., 1988, Transplantation 46:825;Johnston et al., 1992, Transplantation 54:573). These data suggest thatsince complement deposition could be observed in a transplantedxenogeneic organ in the absence of Ig deposition, mechanisms leading torejection may be triggered even if natural xenoreactive antibodies areneutralized or removed from recipient serum.

2.2. αGAL EPITOPE

In recent years, much attention has been focused on defining themolecular structures that are recognized by xenophilic naturalantibodies, leading to activation of the complement cascade andeventually, to hyperacute rejection. Most evidence now points to theoligosaccharide epitope Galα1-3Gal ("αGal") as the major target ofxenoreactive natural antibodies. Humans and old world monkeys do notexpress the αGal epitope because they lack a functional gene encodingthe enzyme α1-3galactosyl transferase that forms the unfucosylated"linear B" epitope Galα1-3Galβ1-4GlcNAc, which in other mammalian cellscauses terminal glycosylation of many glycoproteins, including thoseexpressed by endothelial cells, leukocytes and red blood cells (Galili,et al., 1993, Immunol. Today 14:480-482). Initial evidence of theimportance of the αGal epitope was provided by studies in whichantibodies from porcine organs perfused by human plasma were eluted andtested for binding to immobilized carbohydrates by an ELISA(enzyme-linked immunosorbent assay). Of the carbohydrates tested, theeluted antibodies were observed to bind only to those carbohydratescontaining α-galactose (Good et al., 1992, Transplant Proc. 24:559-562).A subsequent study examining the cytotoxic effect of human and baboonserum on a pig cell line has shown that carbohydrates containing aterminal α-galactose can neutralize cytotoxicity (Neethling et al.,1994, Transplantation 57:959-963). Additionally, Collins et al., haveshown that expression of the Galα1-3Gal antigen in donor organs may besufficient to bring about the immunological reactions leading tohyperacute xenograft rejection and also that removal of Galα1-3Gal fromporcine cells eliminates the binding of 70-80% of xenoreactive antibody(Collins et al., 1994, Xenotransplantation 1:36-46; Collins et al.,1995, J. Immunol. 154:5500-5510).

Recently, the antigenic glycolipid in pig kidney endothelial cells hasbeen identified as a pentasaccharide consisting ofGalα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide (Samuelsson et al., 1994,Immunological Rev. 141:151-168). A study using ELISA and in vitroimmunosorbent assays to compare the ability of this αGal pentasaccharideto bind human anti-pig antibodies with that of the αGal disaccharide(Galα1-3Gal) or the αGal trisaccharide (Galα1-3Galβ1-4GlcNAc) hasindicated that human anti-αGal antibodies are polymorphic, and thatimmunoadsorbents containing these αGal oligosaccharides may be capableof removing anti-αGal activity albeit ineffectively (Goldberg et al.,1995, Transplant Proc. 27:249-250). To date, experiments investigatingthe ability of oligosaccharides containing the αGal epitope toneutralize xenoreactive antibody have been limited to ex vivohemagglutination, ELISA, and cytotoxicity assays. These limited timeframe experiments have demonstrated that the αGal disaccharide(Galα1-3Gal) and αGal trisaccharide (Galα1-3Galβ1-4 GlcNAc) areeffective in neutralizing the anti-αGal antibody in vitro and that theαGal trisaccharide is ten times more effective than the αGaldisaccharide. (Neethling et al., 1996, Transplantation International9:98-101.)

To date, αGal oligosaccharides longer than the αGal tetrasaccharide havenot been tested individually. Further, none of the Galα1-3Galoligosaccharides have been tested in vivo for the ability to blockanti-αGal binding. The ex vivo experiments lack the complexity of theorgan xenograft system in vivo and therefore do not contain othervariables that might participate in the process of rejection in vivo.Additionally, these experiments have been performed using human serumdevoid of cells and it is unclear what role respective pathways play ininitiation of the hyperacute rejection, but it is likely that no singlepathway alone is entirely responsible. For example, the solublecarbohydrate melibiose (Galα1-6Glu) is a disaccharide similarstructurally to Galα1-3Gal that has been shown in vitro to compete withnatural αGal epitopes for human Ig binding, however, in vivoadministration of this composition has failed to prevent hyperacuterejection and has been found to be cytotoxic to other tissue (Ye et al.,1994, Transplantation 58:330-337). Thus in the absence of evidence tothe contrary, hemagglutination and cytotoxicity results cannotreasonably be expected to be predictive of successful xenograftengraftment. The key experiment for modeling human xenotransplantation,the grafting of pig organs into old world monkeys, until now, has notbeen performed.

While scientific data indicates that most human xenoreactive IgM andsome human xenoreactive IgG is specific for the Galα1-3Gal epitope, somehuman xenoreactive natural antibodies directed against otherdeterminants may also be responsible for hyperactive rejection, assuggested by Parker et al. (1995, Transpl. Immunol. 3:181-191),Lesnikoski et al. (1995, Xenograft endothelial host-mononuclear cellactivation and cytokine expression during rejection of pig to baboondiscordant xenografts. Abstracts of the XVth World Congress of theTransplantation Society. Transplantation Proceedings) Ye et al. (1994,Transplantation 58:330; and Collins et al., 1994 Xenotransplantation1:36). Thus far, Galα1-4Gal, Galβ1-3GalNAc, and SO4-3Gal, three otherpig carbohydrate specificities to which humans have natural antibodies,have been identified (Holgersson et al., 1990, J. Biochem. 108:766;Holgersson et al., 1991, Glyconj. J. 8:172; and Good et al., 1992,Transplant Proc. 24:559). This possibility is also suggested by the factthat other species such as the pig, goat, dog, rat, etc., which do notproduce anti-Galα1-3Gal antibodies have xenoreactive natural antibodieswhich presumably recognize other structures (Cameron et al., 1983, J.Surg. Oncol. 22:157-163; Hammer, C. 1989, 21:522-523). Anti-pigantibodies that bind to the protein components on the surface of pigcells have been reported (Tuso et al., Presentation at the AmericanSociety of Transplant Surgeons, 12th Annual Meeting in Houston, May17-19, 1993). It is possible that antibody dependent and othermechanisms are operating through these epitopes independent of the αGalepitope and would be resistant to its inhibitors.

One of the principal concerns of intravenous carbohydrate therapy inxenotransplantation is whether effective xenograft rejection inhibitioncan be achieved at acceptable non-toxic levels of oligosaccharide. Asdiscussed supra, while ABO-incompatible rejection has been inhibitedsuccessfully using intravenous soluble carbohydrates as antibodyinhibitors (Cooper et al., 1993, Transplantation 56:769-777), thisapproach has been unsuccessful in previous pig/primatexenotransplantation where the necessary concentrations of the anti-αGalantibody inhibitor melibiose (Galα1-6 Glc) proved highly toxic.Anti-αGal antibodies are known to bind to αGal oligosaccharides withrelatively low affinity (Parker et al., 1995 Transplant Immunology3:181-191; Parker et al., 1994, J. Immunology 153:3791-3803). This lowaffinity is believed to necessitate a high concentration ofoligosaccharide in the recipient's blood in order to block the bindingof circulating anti-αGal antibodies to the transplanted organ and mayresult in side effects due to the high concentrations of carbohydrate(see, e.g., U.S. Pat. No. 5,560,911). This low binding affinity is alsothought to possibly have an adverse impact on extracorporealimmunoaffinity treatment by making the removal of anti-αGal antibodiesrelatively inefficient (see, e.g., U.S. Pat. No. 5,560,911).

A complicated series of multiple overlapping events contribute to therecognition of vascularized discordant grafts, including binding ofpreformed natural antibodies, complement activation, activation of thecoagulation cascade and endothelial cell activation. Due to thesemultiple overlapping events, the possible involvement of theantibody-independent alternative pathway, the potential involvement ofxenoantibodies other than those specific for the αGal epitope, and thelow binding affinity of anti-αGal antibodies for αGal oligosaccharides,one would not reasonably expect that treatment with αGaloligosaccharides would be effective in neutralizing anti-αGal antibodiesand even if they were, that such neutralization and/or removal ofanti-αGal antibodies would be sufficient to overcome HAR or to attenuatexenograft rejection.

2.3. CURRENT APPROACHES TO OVERCOME XENOGRAFT REJECTION

Other approaches for overcoming HAR are also being explored. Theseapproaches generally aim to genetically engineer pigs so that they donot trigger the rejection reaction, by for example, expressing elevatedlevels of complement regulatory sequences on the surface of endothelialcells (Langford et al., 1993, Abstract #56, Second InternationalCongress on Xenotransplantation, Cozzi et al., 1993, Abstract #57,Second International Congress on Xenotransplantation), expressingfucosyl transferase that competes with α-galactosyltransferase foracceptors and fucosylates the acceptor moiety (Sandrin et al., 1996,Xenotranspl. 3:134-140 and Sandrin et al., 1995, Nature Med.1:1261-1267), or by knocking out the gene encoding α-galactosyltransferase. An alternative approach attempts to tolerize prospectivehuman recipients to pig tissues, by for example, inducing immunologicalchimerism (see e.g., Tanaka et al., 1993, Abstract #122, SecondInternational Congress on Xenotransplantation; Zeng et al., 1992,Transpl. Proc. 24:641; Zeng et al., 1992, Transplantation 53:277;Ricordi et al., 1992, Surgery 112:327; Ildstad et al., 1992,Transplantation 53:815; and Ildstad et al., 1992, J. Exp. Med. 175:147).Additionally, the use of human anti-xenograft, anti-idiotypic antibodieshas also been proposed as a means by which to inhibit acutecomplement-mediated cytotoxicity (see U.S. Pat. No. 5,560,911,). Todate, no one has successfully been able to achieve clinicallysignificant attenuation of xenograft rejection in vivo, and it isunlikely that these other approaches will succeed in the near future.

2.4. RETARGETING OF HOST EFFECTOR MECHANISMS

Methods of retargeting host effector mechanisms to targets oftherapeutic interest using bispecific agents have been reported in theart (see e.g., Meeting Report of the Second International Conference onBispecific Antibodies and Targeted Cellular Toxicity, February 1991,Immunol. Today, 12(2):51-54). At the most basic level, any therapeuticantibody can be described as a bispecific agent that retargets hostdefense mechanisms to a chosen target. The antigen-binding "front" endof an antibody binds the antigenic epitope on a tumor cell, for example,and the Fc "tail" serves to attract and deliver host effectormechanisms, specifically complement or cells that possess receptors forthe tail region of the antibody. These receptors, FcR, come in severalvarieties which bind different antibody populations and are expressed ondifferent cell types (macrophages, neutrophils, natural killer, or NKcells, etc.). The host effector mechanisms are the ones that do thedamage: complement forms a "membrane attack complex" comprised ofcomponents C5b-C9, which lyses the target cell, while FcR⁺ cells destroytarget cells by either phagocytosis or by perforation of their membranewith lytic molecules (e.g., perforin, granzyme). Many strategies forretargeting cytotoxic cells have involved the use of bispecificconjugates of antibodies in which one antibody is directed against thecytotoxic cell receptor involved in lysis, while the second antibody isdirected against a target cell structure, such as, for example, a tumoror viral antigen (see e.g., Donohue et al, 1990, Cancer Res.50:6508-6514; Van Dijk et al., 1989, Int. J. Cancer, 44:738-743; andSegal et al., 1988, Princess Takamatsu Symp. 19:323-331). Theadministration of chemically cross-linked bispecific monoclonalantibodies reacting with CD3 on T-cells and with cell-surface antigensselectively expressed by tumor cells has been shown to targetT-lymphocytes to neoplastic cells and to significantly decrease thegrowth of an established tumor in vivo (Garrido et al., 1990, Canc. Res.50:4227-4232).

Recently, Pouletty has described a conjugate for inactivating targetcells in a mammalian host which consists of a ligand for the target celland a component that binds an endogenous cytotoxic effector system(European patent No. EPO 510949, issued Jan. 22, 1997). Pouletty furtherdiscloses methods for using these compounds to inactivate a target cell.Pouletty also describes the use of saccharides, such as the blood groupA-trisaccharide antigen, as the effector-binding component of theconjugate and discloses the in vitro lysis of CTL-L2 lymphocytes afterincubation with an IL2-blood group A conjugate and human serumcontaining anti-blood group A antibodies. However, Pouletty does notdescribe or suggest the use of αGal oligosaccharides as the effectorbinding component of the conjugate. Nor does Pouletty describe orsuggest harnessing the pre-existing anti-αGal antibodies in the humanserum as an effector agent for complement-mediated lytic attack. Indeed,Pouletty does not even disclose whether the blood group A antigeneffector-binding component of the conjugate is effective in binding theendogenous effector system to form a cell inactivating complex in vivo.

Recently, Lussow et al. have described using an IL2-fluoresceinconjugate to target anti-fluorescein antibodies to activated T cells,and thereby deplete the targeted cells in vivo (1996, TransplantationProc., 28:571-572). Lussow et al. disclose that a IL2-fluoresceinconjugate component ratio of 1:1 is critical for preventing clearance ofthis conjugate from the circulation. While Lussow et al. suggest usingthe αGal epitope as the effector binding component of the conjugate toharness the hyperactive rejection response and redirect this response todesired targets, this reference does not teach whether anyoligosaccharides, let alone oligosaccharides containing αGal epitopes,can actually bind an endogenous effector system in vivo. Further, asdiscussed above, anti-αGal antibodies are known to bind to αGaloligosaccharides with relatively low affinity and it is doubtful that amonovalent αGal oligosaccharide would bind anti-αGal antibodies in vivo(see e.g., Parker et al., 1994, J. Immunology 153:3791-3803; Parker etal., 1995, Transplant Immunology 3:181-191). Accordingly, Lussow et al.,does not provide a reasonable expectation that αGal oligosaccharide-cellligand conjugates could successfully be used to effectuatecomplement-mediated lytic attack of targeted cells in vivo.

Citation of a reference hereinabove shall not be construed as anadmission that such reference is prior art to he present invention.

3. SUMMARY OF THE INVENTION

The present invention relates to methods for attenuating xenograftrejection of transplant tissue between discordant species. Xenograftrejection is thought to be mediated, in part, by xenoreactive antibodiesto a Galα1-3Gal motif ("αGal") containing carbohydrate ligand found onthe endothelium of xenogeneic organs to initiate complement activation.The invention is based, in part, on the discovery that administration ofoligosaccharides comprising the αGal motif, is sufficient to attenuatexenograft rejection in vivo. Additionally, the Inventors have made thesurprising discovery that the oligosaccharide compositions of theclaimed invention are effective at competitively inhibiting binding ofanti-αGal antibodies to xenografts under physiological conditions atnontoxic concentrations and that other xenoantibodies do not play adeterminative role in xenograft survival. Accordingly, the presentinvention relates to methods for inhibiting anti-αGal antibody bindingto donor organ endothelium, by administering an effective amount of apharmaceutical composition comprising an amount of an αGaloligosaccharide, or a pharmaceutically acceptable derivative thereof,sufficient to bind anti-αGal antibodies so as to competitively inhibitbinding of these antibodies to donor organ endothelium, and to therebyattenuate xenograft rejection and/or trauma resulting from anti-αGalantibody-mediated complement activation.

Also described are methods for suppressing lymphocytes bearing anti-αGalidiotypes and for predicting the severity of xenotransplant rejection inhuman recipients. In preferred embodiments, the administration ofcompositions comprising αGal oligosaccharides or pharmaceuticallyacceptable derivatives thereof, that are multivalent or associated withor conjugated to cytocidal agents, targets destruction of B cellsbearing surface exposed anti-αGal immunoglobulins (or idiotypes).

Human serum may have up to 1% of IgG and IgM with specificity for theαGal epitope (Galili et al., 1993, Immunol. Today 14:480-482). In oneembodiment, compositions comprising αGal oligosaccharides, orpharmaceutically acceptable derivatives thereof, target anti-αGalantibodies to tissue or cell types having a distinguishing surfacemarker. This method comprises administering a pharmaceutical compositioncomprising an amount of an αGal oligosaccharide, or a pharmaceuticallyacceptable derivative thereof, associated with or conjugated to a ligandfor a specific cell-surface marker so as to target anti-αGal antibodiesto tissue or cells bearing this marker. This method may be applied inhumans or old world monkeys to target complement-mediated lyticdestruction or phagocytosis of any cells, viruses, or tissue expressingthe distinguishing surface marker, including, but not limited to, cellsor tissue responsible for autoimmunity disorders, viral diseases,parasitic diseases or immunosuppression.

The αGal oligosaccharide composition may be administered alone or incombination with other agents useful in attenuating xenograft rejection,including conventional nonspecific immunosuppressive agents, such as,for example, cyclosporine, cyclophosphamide, methylprednisolone,prednisone, and azathioprine. In a further embodiment, thepharmaceutical compositions comprise an anti-inflammatory and/orantibiotic and/or anti-thrombolytic.

It is a primary object of this invention to provide a method andassociated compositions for attenuating xenograft rejection or toalleviate trauma caused by anti-αGal antibody triggeredcomplement-mediated lytic attack by interfering with complementactivation resulting from anti-αGal antibody binding to cell surfaces,in particular donor organ endothelium. Accordingly, the pharmaceuticalcompositions of the invention may be administered alone, together with,or in seriatim with other therapy regimens directed toward reducingbinding of xenoreactive antibody in host serum, preferably anti-αGalantibody to donor organ cells or tissue.

Xenoantibodies may be neutralized in vivo, by additional techniqueswhich include, but are not limited to, the administration of humananti-animal anti-idiotypic antibodies, and ex vivo by additionaltechniques which include, but are not limited to, extracorporealimmunoaffinity treatment with human anti-animal idiotypic antibodies,plasmapheresis, perfusion of recipient blood through donor organs, cellsor tissue, and exposure to αGal oligosaccharide compositions of theinvention.

In addition to therapeutic methods, the invention also relates topharmaceutical compositions comprising an amount of αGaloligosaccharide, or a pharmaceutically acceptable derivative thereof,sufficient to competitively inhibit binding of anti-αGal antibodies todonor organ endothelium, and a pharmaceutically acceptable carrier. Theinvention further relates to pharmaceutical compositions comprising anamount of αGal oligosaccharide, or a pharmaceutically acceptablederivative thereof, effective in targeting anti-αGal antibodies tomarked cells or tissue, and a pharmaceutically acceptable carrier.

The compositions of the invention may include any oligosaccharidecomprising the αGal motif (i.e., Galα1-3Gal), including but not limitedto, the αGal oligosaccharides Galα1-3Gal (αGal disaccharide),Galα1-3Galβ1-4(Glc or GlcNAc) (αGal trisaccharide),Galα1-3Galβ1-4GlcNAcβ1-3Gal (αGal tetrasaccharide) andGalα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc (αGal pentasaccharide) whichcorresponds to the antigenic glycolipid in pig kidney endothelial cells,or any combination thereof. Other embodiments of the invention aredirected to pharmaceutically acceptable derivatives of αGaloligosaccharides.

The αGal oligosaccharides of the invention may be monovalent ormultivalent and may comprise one or multiple αGal oligosaccharides. Inspecific embodiments, the monovalent or multivalent αGal compositions ofthe invention comprise the αGal disaccharide, trisaccharide,tetrasaccharide and/or pentasaccharide, corresponding to the antigenicglycolipid in pig kidney endothelial cells, or a pharmaceuticallyacceptable derivative thereof. The αGal oligosaccharide component isoptionally associated with or conjugated to biologically inert moleculesto enhance (or reduce) stability or biological half-life, reducetoxicity, target cells or tissue and/or to temporarily mask the αGalepitope.

In a particular embodiment of the invention described by way of examplein Section 6.7, the ability of the αGal pentasaccharideGalα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc to effectively neutralize anti-αGalantibody in vivo and to thereby attenuate xenograft rejection of a pigheart in a baboon recipient is disclosed.

3.1. DEFINITIONS

As used herein, "Gal" refers to galactose; "Glc" refers to glucose; and"GlcNAc" refers to N-acetylglucosamine.

As used herein, the term "αGal oligosaccharide" refers both to compoundscomprising the αGal motif (Galα1-3Gal) and to such compounds associatedor conjugated with an αGal oligosaccharide as described herein. αGaloligosaccharides are defined herein as organic compounds comprising twoor more saccharide moieties in which a galactose moiety is covalentlyjoined by an α1-3 glycosidic linkage to another galactose moiety. αGaloligosaccharides may be referred to with respect to the number ofsaccharide units corresponding to the antigenic glycolipid in pig kidneyendothelial cells, i.e., a disaccharide comprises Galα1-3Gal; an αGaltrisaccharide comprises Galα1-3Galβ1-4GlcNAc; an αGal tetrasaccharidecomprises Galα1-3Galβ1-4GlcNAcβ1-3Gal; and an αGal pentasaccharidecomprises Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc.

As used herein, "hyperacute rejection" refers to rapid graft rejection,beginning minutes after implantation, and which is mediated bypre-existing antibodies to the graft.

As defined herein, a "xenograft" may be an organ, tissue, aggregates ofcells, or cells, collectively referred to herein as "tissue." The tissuemay be selected from any appropriate tissue of the body of the tissuedonor. These tissues include, but are not limited to, heart, kidney,lung, islet cells, liver, bowel and skin cells.

As used herein, the phrase "attenuation of xenograft rejection" means toinhibit or interfere with processes leading to hyperacute rejection of axenotransplant. The treatment is considered therapeutic if there isinhibition, delay, or reduction of symptoms associated with hyperacuterejection such as ischemia, thrombosis, myocardial congestion and tissuenecrosis.

As used herein, the terms "marked cells" or "cells containing a surfacemarker" refer to cells expressing a molecule on their surface thatallows for targeting of a composition of the invention to the surface ofthe cells.

The term "pharmaceutically acceptable carrier" refers to a carriermedium that does not interfere with the effectiveness of the biologicalactivity of the active ingredient, is chemically inert and is not toxicto the subject to which it is administered.

As used herein the term "pharmaceutically acceptable derivative" refersto any homolog or analog corresponding to αGal oligosaccharides asdescribed in Section 5.1. infra, which binds anti-αGal antibodies and isrelatively non-toxic to the subject to which it is delivered.

The term "therapeutic agent" refers to any molecule, compound ortreatment, preferably an anti-inflammatory, anti-thrombolytic and/orantibiotic, that assists in reducing untoward effects resulting fromxenotransplantation.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B In vitro mouse laminin ELISA evaluation of soluble αGaloligosaccharides as neutralizers of anti-αGal antibodies. Immobilizedmouse laminin serves as a capture ligand for anti-αGal antibodies. FIG.1A and FIG. 1B show the extent of anti-αGal IgG and IgM antibodyneutralization as a function of αGal oligosaccharide or sucroseconcentration (μM), respectively. Diamonds represent αGal disaccharide;circles represent αGal trisaccharide; triangles represent αGalpentasaccharide; and squares represent sucrose. The binding of anti-αGalantibodies is inhibited to different extent by αGal oligosaccharides ofdifferent lengths. The non-αGal oligosaccharide tested does not inhibitantibody binding.

FIGS. 2A-2B In vitro immobilized αGal oligosaccharide ELISA analysisevaluation of soluble αGal oligosaccharides as neutralizers of anti-αGalantibodies. FIG. 2A and FIG. 2B show the extent of anti-αGal IgG and IgMantibody neutralization as a function of soluble αGal oligosaccharideconcentration (mM), respectively. Squares represent Galα1-3Gal; shadeddiamonds represent Galβ1-4Gal; circles represent Galα1-3Galβ1-4Gal;diamonds represent Galα1-3LacNAc; triangles representGalα1-3Galβ1-4Galα1-3Gal; inverted triangles represent Galα1-3 LNnT(αGal pentasaccharide); and shaded circles represent sucrose.

FIGS. 3A-3B In vitro cytotoxicity evaluation of αGal oligosaccharides asneutralizers of anti-αGal antibodies. The extent of pig kidney cell linePK-15 cytotoxicity is shown as a function of soluble αGaloligosaccharide (CHO) concentration (mM). Squares represent αGaltrisaccharide (Galα1-3Galβ1-4 Gal) and circles represent αGalpentasaccharide (Galα1-3Galβ1-4Galα1-3Galβ1-4Glc). Cytotoxicity isinhibited slightly better by the αGal trisaccharide than by the αGalpentasaccharide.

FIG. 4 Cytotoxicity of serum from a baboon receiving αGalpentasaccharide (αGal-LNnT) intravenously. Shaded circles representcytotoxicity of serum as recovered from baboon against mouse endothelialcell line MAE. Other lines indicate varying concentrations ofexogenously added αGal-pentasaccharide into cytotoxicity assay. Addedconcentrations of the αGal pentasaccharide are depicted as follows: 2.0mM, open circle; 1.0 mM, ×; 0.5 mM, diamond; and 0.25 mM, open square.This assay shows that α-Gal pentasaccharide compositions of greater than1 mM inhibit cytotoxicity, and that αGal-pentasaccharide by itself fullyinhibits MAE cell cytotoxicity (similar results were obtained with pigPK15 cells).

FIGS. 5A-5B Blood αGal pentasaccharide concentration and inhibition ofserum cytotoxicity. FIG. 5A, inhibition of cytotoxicity of serum frombaboon xenotransplant #1 receiving αGal pentasaccharide intravenously asa function of the plasma concentration of αGal pentasaccharide. FIG. 5B,inhibition of serum cytotoxicity of baboon serum of baboonxenotransplant #2 receiving αGal pentasaccharide intravenously as afunction of the plasma concentration of αGal pentasaccharide. The lineis αGal pentasaccharide concentration in baboon plasma. Crossesrepresent % inhibition of cytotoxicity of mouse endothelial cell line,MAE. Circles represent % inhibition of cytotoxicity of pig kidneyendothelial cell line, PK-15.

FIGS. 6A-6B Depletion of anti-αGal antibodies from human serum bypassage over αGal-sepharose. FIG. 6A, inhibition of serum cytotoxicityof pooled human serum lot #1 passaged over αGal trisaccharide (shadedsquare) and αGal pentasaccharide (open circle). FIG. 6B, inhibition ofserum cytotoxicity of pooled human serum lot #2 passaged over αGaltrisaccharide (shaded square), αGal pentasaccharide (open circle), andglucose (open triangle).

5. DETAILED DESCRIPTION OF THE INVENTION

The invention relates to methods for attenuating xenograft rejection inmammals, including humans and old world monkeys, which comprisesadministering an amount of an αGal oligosaccharide, or apharmaceutically acceptable derivative thereof, effective inneutralizing or removing anti-αGal antibodies. The invention alsorelates to methods of identification, isolation and suppression oflymphocytes bearing anti-αGal idiotypes. The invention additionallyencompasses administering an effective amount of an αGaloligosaccharide, or a pharmaceutically acceptable derivative thereof, totarget anti-αGal antibody mediated complement activation to cells ortissues containing distinguishing cell-surface markers. The inventionfurther comprises pharmaceutical compositions that may be used in thepractice of the invention to attenuate xenograft rejection and/or targetanti-αGal directed complement-mediated lytic attack.

The present method provides treatment for attenuating xenograftrejection in humans and old world monkeys, such as for example, in pigto human discordant xenografting. Specifically, the invention providesαGal oligosaccharide compositions, or pharmaceutically acceptablederivatives thereof, capable of competitively inhibiting the binding ofpreformed host anti-αGal antibodies to xenografts and thereby preventingactivation of anti-αGal directed complement-mediated lytic attack of thetransferred tissue which leads to hyperacute rejection of the xenograft.The compositions of the invention may be administered alone or incombination with other therapeutic agents, such as, for example,classical immunotherapeutic agents, anti-inflammatories, and/orantibiotics. The invention also encompasses the use of combinations ofdistinct αGal oligosaccharides, e.g., αGal trisaccharide in combinationwith αGal pentasaccharide.

The pharmaceutical compositions of the invention may be administeredalone, together with, or in seriatim with other therapy regimensdirected toward reducing binding of xenoreactive antibodies to donororgan cells or tissue. An example of an in vivo technique that may beused in combination with infusion and/or ex vivo column depletion withαGal oligosaccharide compositions of the invention includes, but is notlimited to, infusion with human anti-animal idiotypic antibodies.Examples of ex vivo techniques that may be used in combination withinfusion and/or ex vivo column depletion with αGal oligosaccharidecompositions of the invention include, but are not limited to,extracorporeal immunoaffinity treatment with human anti-animal idiotypicantibodies, plasmapheresis and perfusion of host blood through donororgans or tissue.

In specific non-limiting embodiments of the present invention detailedin the examples sections infra, anti-αGal antibody neutralization andcytotoxicity studies performed on serum drawn from baboons infused withthe αGal pentasaccharide of the invention, are described. Attenuation ofxenograft rejection in baboon-pig heart recipients that have beeninfused with αGal pentasaccharide is also described.

The Inventors have discovered that administration of the αGaloligosaccharides of the invention is effective in achieving significantattenuation of xenograft rejection in. vivo. The methods disclosedherein present the first known successful use of oligosaccharide therapyto attenuate xenograft rejection.

Although described herein with specific reference to pigs, the samecompositions and methodology can be used to overcome hyperacuterejection of xenografts from other donor species having a functionalα1-3 galactosyl transferase by recipients, such as old world monkeys orhumans which do not have a functional α1-3 galactosyltransferase.

5.1. αGAL OLIGOSACCHARIDE COMPOSITIONS

The invention provides pharmaceutical compositions comprising αGaloligosaccharides or pharmaceutically acceptable derivatives thereof,that competitively inhibit binding of anti-αGal antibodies toxenografts. By binding to these antibodies, oligosaccharide compositionsof the invention are able to neutralize, remove and/or target anti-αGalantibodies to specific tissue or cell types.

αGal oligosaccharides of the invention include those saccharidecompositions comprising two or more saccharide moieties in which agalactose moiety is joined by an α1-3 glycosidic linkage to anothergalactose moiety. In preferred embodiments the αGal motif (Galα1-3Gal)is located within 15, 10, 5, 4, 3, 2 or 1 saccharide unit(s) from thenon-reducing end of the oligosaccharide. In a most preferred embodiment,the αGal motif represents the terminus (i.e., the non-reducing end) ofthe oligosaccharide. The αGal oligosaccharides may comprise one, or aplurality of αGal motifs. For example, the αGal oligosaccharide may be abranched carbohydrate having multiple terminal Galα1-3Gal residues or alinear oligosaccharide containing both terminal and internal Galα1-3Galresidues.

In preferred embodiments, the αGal oligosaccharides of the inventioncomprise a saccharide sequence corresponding to the antigenic glycolipidexpressed on pig kidney endothelial cell membranes. Such αGaloligosaccharides include, but are not limited to Galα1-3Gal;Galα1-3Galβ1-4GlcNAc; Galα1-3Galβ1-4GlcNAcβ1-3Gal; andGalα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc. In a most preferred embodiment, theαGal oligosaccharide is the pentasaccharide,Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc.

The compositions of the invention also include pharmaceuticallyacceptable derivatives of αGal oligosaccharides. Such derivativesinclude but are not limited to salts, pyran ring derivatives,multivalents and conjugates or associations with a αGal oligosaccharide.

In one embodiment, the pharmaceutically acceptable derivatives of αGaloligosaccharides are sulfate substitutes and salts thereof. Suitablecations include alkali metals, alkaline earth metals or ammonium. Anyknown suitable pharmaceutically acceptable cations may be used,including the cations of conventional non-toxic salts including a metalsalt such as an alkali metal salt (e.g., sodium salt, potassium salt,etc.) or an alkaline earth metal salt (e.g., calcium salt, magnesiumsalt, etc.), an ammonium salt, an organic base salt (e.g.,trimethylamine salt, triethylamine salt, pyridine salt, picoline salt,dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, etc.), a saltwith an amino acid (e.g., arginine salt, aspartic acid salt, glutamicacid salt, etc.), and the like.

The pharmaceutically acceptable derivative of the invention may includepyran ring variants in which the oxygen of one or more pyran rings isreplaced by another heteroatom. For example, in one embodiment the αGaloligosaccharide derivative comprises an αGal aza sugar in which theoxygen of one or more of the pyran rings is replaced with a nitrogen, toform a piperidine ring system. In another embodiment, the oxygen of oneor more of the pyran rings is replaced with a sulfur, to form atetrahydrothiopyran ring system.

In alternative embodiments, the pharmaceutically acceptable derivativeof the invention comprises an αGal oligosaccharide associated orconjugated with a blocking, or masking, agent capable of blockingxenoantibody binding to the αGal oligosaccharide for a predeterminedlength of time. Suitable blocking agents are known to those in the artand may be removed by natural processes or pharmacological intervention,thereby exposing the αGal epitope. Masking may be achieved by techniquesknown in the art, including but not limited to, polyacetylation of theαGal oligosaccharide so that serum or administered esterases wouldde-acetylate and expose the αGal epitope, and capping of one or moreterminal αGal motifs with another monosaccharide to alter its antigenicnature, which would be hydrolyzed by a natural or administeredglycosidase.

The αGal oligosaccharide of the invention may be associated orconjugated with other molecules. These molecules may be macromolecularcarrier groups including, but not limited to, lipid-fatty acidconjugates, polyethylene glycol, protein or carbohydrate. The associatedor conjugated molecule may also provide bifunctionality to the αGaloligosaccharide by, for example, targeting αGal oligosaccharides topredetermined tissue or cell types. The association or conjugationbetween the αGal oligosaccharide and the other molecule may be theresult of a direct interaction, such as for example, through a chemicalbond or ionic interaction, or alternatively, the association orconjugation with the other molecule may be through a linking group. Thelinking group can be any group known in the art which serves to link theαGal oligosaccharide, or pharmaceutically acceptable derivative thereof,with the other molecule. Suitable linking groups include saccharides,oligosaccharides, peptides, proteins, C₂₋₂₀ alkyl, oxyalkylene chains orany other group, which does not inhibit the ability of the αGaloligosaccharide component of the composition to bind anti-αGalantibodies. The ability of αGal oligosaccharide components of thecomposition to bind anti-αGal antibodies may be determined applyingassays described in Sections 6.3, 6.4 and 6.5 as well as those known inthe art.

The αGal oligosaccharide or pharmaceutically acceptable derivative ofthe invention may be monovalent or multivalent. Competitive inhibitionis typically enhanced by increased valence, as once the first contacthas been made, the probability of subsequent contact taking place isfavored thermodynamically. The use of multivalents is especially usefulin blocking low affinity events where high avidity can compensate. Suchis the case for anti-αGal antibodies which, like most anti-carbohydrateantibodies, especially IgM, are of relatively low affinity. The directcorrelation between competitive inhibition and valence is demonstratedby BSA glycoconjugates which provide a model for multivalence and whichinhibit binding with IC₅₀ values in the μM range, as opposed tomonovalents which inhibit in the mM range (Simon et al., personalobservation). In a specific embodiment, the composition comprisesmultivalent αGal oligosaccharides or αGal oligosaccharide structures toincrease the potency and/or biological half-life of the pharmaceutical.In particular embodiments, the αGal oligosaccharide of the invention isdivalent, trivalent, tetravalent, pentavalent, heptavalent ordecavalent. In one embodiment, an αGal oligosaccharide (e.g., the αGaltetrasaccharide or αGal pentasaccharide) is found in multiple copies ona compound for use in the invention. In another embodiment, more thanone αGal oligosaccharide (e.g., the tetrasaccharide and pentasaccharide)are found in single or multiple copies on a compound for use in theinvention. In another embodiment, the oligosaccharide orpharmaceutically acceptable derivative of the invention comprises 2, 3,4, 5, 10, 20 or 30 αGal motifs on one molecule or is polyvalent.

Multivalent carbohydrates can be prepared using methods known in the artto prepare a branching complex carbohydrate, which conceptuallyresembles a tree or brush in which each branch or bristle contains aanti-αGal oligosaccharide motif. In preferred embodiments, each branchor bristle is terminated by an αGal motif. Alternatively, monovalentcarbohydrates can be associated covalently or noncovalently with apolymer using techniques known in the art (See e.g., Langer et al.,International Patent Publication No. WO 94/03184, published Feb. 17,1994, which is herein incorporated by reference in its entirety). TheαGal oligosaccharide or pharmaceutically acceptable derivative thereofmay be bound directly or through a linking group to the polymer usingknown techniques so as to produce a conjugate in which more than oneindividual molecule of the oligosaccharide is covalently attached.Suitable linking groups include, but are not limited to saccharides,oligosaccharides, peptides, proteins, C₂₋₂₀ alkyl, oxalkylene chains orany other group which does not prevent the anti-αGal antibody binding toαGal oligosaccharide. Suitable polymer supports are compounds withmultiple binding sites to the reducing end saccharide or to a terminalend of the linking group which is not bound to the reducing endsaccharide, or with multiple binding sites to the C₁, glycosidic oxygenof a glucose or N-acetylglucosamine residue. Suitable polymers are knownin the art and include, but are not limited to, a polyol, apolysaccharide, avidin, lipids, lipid emulsions, liposomes, a dendrimer,human serum albumin, bovine serum albumin, a protein, polylysine,dextran, a glycosaminoglycan, cyclodextrin, agarose, sepharose, andpolyacrylamide.

Multivalent αGal oligosaccharide compositions may be used to neutralizeanti-αGal antibodies and/or to target complement-mediated lytic attackto B lymphocytes bearing anti-αGal idiotypes. The construct needed toachieve this effect possesses two or more αGal epitopes on one molecule,or is multivalent. While not wishing to be bound to theory, thesecompositions would deplete B lymphocytes bearing anti-αGal idiotypes bya mechanism in which one αGal group would be bound by the B-cell'ssurface immunoglobulin, while the others would be displayed outward,serving as surface ligands for the circulating anti-αGal antibodies.Alternatively, multivalent αGal constructs may down-regulate theproduction of anti-αGal antibodies by the receptor cross-linking effectdescribed by Dintzis et al. (1990, Eur. J. Immunol., 20:229; U.S. Pat.No. 5,370,871; U.S. Pat. No. 5,126,131; 1976, PNAS, 73:3671-3675; 1990,Immunol. Rev. 115:243-250). In an alternative embodiment, one or more ofthe αGal epitopes are "masked" by a labile group using known techniques.According to this embodiment, exposed αGal epitopes bind theB-lymphocyte surface immunoglobulin and the masking group is removedgradually by natural or pharmacological intervention, exposing themasked αGal group(s), and bringing about complement-mediated lysis ofthe B-lymphocytes. The αGal oligosaccharides or pharmaceuticallyacceptable derivatives of the invention may be masked by masking groupsknown in the art, including but not limited to acetyl groups that may bedeacetylated by serum esterases and thereby expose the αGal epitope; andcapping of the αGal motif with a monosaccharide or polysaccharide thatalters the antigenic nature of the αGal motif, and which may behydrolyzed by a natural or administered glycosidase. The ability ofmasking groups to alter the antigenic nature of the αGal motif and forunmasking to expose the epitope may be routinely determined applying theassays described in Sections 6.3, 6.4 and 6.5.

In another embodiment, the αGal oligosaccharide is chemically linkedeither directly or through a linker to a cytocidal agent. The αGaloligosaccharide may be chemically linked to any cytocidal agent, usingknown techniques. Such cytocidal agents include, but are not limited to,toxins (e.g., ricin A, Pseudomonas exotoxin) or cytotoxic drugs (e.g.,cytosine arabinoside, daunorubicin). αGal oligosaccharide/cytocidalagent compositions of the invention may be administered to targetcytocidal attack of B lymphocytes bearing anti-αGal idiotypes.

The αGal oligosaccharide, or pharmaceutically acceptable derivative ofthe invention may be associated (e.g., ionic interaction) or conjugated(e.g., covalent linkage) with a ligand for a cell-surface molecule so asto target anti-αGal antibodies to tissue or cells expressing thesemolecules. Such oligosaccharide-ligand combination may be through thedirect interaction of the oligosaccharide and ligand or indirectly usinglinker means known in the art. The oligosaccharide/ligand combinationmay be generated by techniques known in the art (See e.g., Stowell andLee, 1980, Advances in Carbohydrate Chemistry, 37:225-281, which isherein incorporated by reference in its entirety) and are generated soas not to inhibit binding of the αGal oligosaccharide to anti-αGalantibodies. The ability of the αGal oligosaccharide/ligand combinationto bind anti-αGal antibody may routinely be determined applying in vitroassays described herein (see Sections 6.3, 6.4 and 6.5) and known in theart. The ability of the αGal oligosaccharide/ligand combination to bindto the cell-surface binding partner of the ligand may be determinedusing techniques known in the art. The ligand component of theoligosaccharide/ligand combination may comprise monoclonal antibody,cell-surface receptor ligand or other homing molecules fortherapeutically significant targets that are known or may routinely beidentified and isolated and/or generated using techniques known in theart. For example, for the generation of monoclonal antibodies seegenerally, Harlow, E., 1988, Antibodies a Laboratory Manual, Cold SpringHarbor, Ed. by Harlow and Lane.

In another preferred embodiment, the αGal oligosaccharide, orpharmaceutically acceptable derivative thereof is associated orconjugated to an autoantigenic peptide for specifically targetinganti-αGal antibodies to the major histocompatibility complex ofautoreactive lymphocytes. In other embodiments, the αGal oligosaccharideor pharmaceutically acceptable derivative thereof is associated orconjugated to dominant auto-antigenic peptide epitopes associated withdiseases. Such epitopes include, but are not limited to, myelin basicprotein peptides in multiple sclerosis, pancreatic islet autoantigenicpeptides on p54 in juvenile (type I or autoimmune) diabetes mellitus,acetylcholine receptor peptides in myasthenia gravis, and collagenpeptides in rheumatoid arthritis.

In another preferred embodiment, the αGal oligosaccharide orpharmaceutically acceptable derivative thereof is conjugated to anantibody (e.g., monoclonal) for targeting complement-mediated lyticattack to tissue or cell types expressing antigen for the antibody.Since these conjugates localize complement-mediated lytic attack ofmarked tissue or cell types, the conjugates provide an alternative cidalmechanism to classical antibody-based chemotherapy. Examples of tissueor cell types that may be targeted for complement-mediated lytic attackinclude tumors which express specific antigens to which antibodies havebeen developed. In preferred embodiments, the αGal oligosaccharide orderivative is conjugated to the minimal antigen-binding region of atumor-specific antibody which is identified and generated usingtechniques known in the art. In other preferred embodiments, the αGaloligosaccharide or derivative is conjugated to single chain antibodieswith binding characteristics equivalent to those of the original tumorspecific monoclonal antibody. Such single chain antibodies may beselected using antigen-driven screening systems known in the art, (Seee.g., McCafferty et al., 1990, Nature, 348:552-554; Clackson et al.,1991, Nature, 352:624-688). These lower molecular weight versions havebetter vascular access and faster renal clearance than complexescontaining the intact monoclonal antibody. Additionally, these truncatedantibodies or single chain antibodies contain a smaller number ofepitopes than the intact monoclonal antibody, and thereby represent amuch weaker immunogenic stimulus when injected into the patient (e.g.,human). An intravenous injection of the single chain antibody ortruncated antibody is, therefore, expected to be more efficient andimmunologically tolerable in comparison with currently used wholemonoclonal antibodies (Norman et al., 1993 Transplant. Proc. 25 Suppl.1:89-93).

In an additional preferred embodiment, the αGal oligosaccharide, orpharmaceutically acceptable derivative thereof, is coupled to a ligandbound by a pathogenic virus or by a virally infected cell. Ligandsencompassed by this embodiment include, but are not limited to,CD4-derived peptide bound by gp120 of HIV (from the D1 domain of CD4 anddistinct from the MHC-binding region (see e.g., Sakihama et al., 1995,PNAS 92:644-648; and Ryu et al., 1994, Structure 2:59-74), peptidesderived from the extracellular domain of chemokine receptors (e.g., CCCKR-5 or fusin) to which the V3 loop of gp120 binds (Choe et al., 1996,Cell 85:1135-1148; Feng et al., 1996, Science 272:872-876), andNeuAcα2-6Galβ1-4Glc ligand for influenza virus, a recessed hemagglutininligand which is conserved among the main serotypes of the virus (Connoret al., 1994, Virology 205:17-23; and Suzuki, Y., 1994, Prog. Lip. Res.33:429-457).

In another preferred embodiment, the αGal oligosaccharide, orpharmaceutically acceptable derivative thereof, is coupled to a ligandthat binds targets located on parasites and may be used in the treatmentof parasitic diseases. Ligands that target parasitic organisms include,but are not limited to, NeuAcα2-3Gal, the ligand for Trypanosoma cruzi(Chagas disease) trans-sialidase, and also for Plasmodium falciparum(malaria) adherence to erythrocytes, as well as Gal/GalNAc-terminatingoligosaccharides that are bound by Entamoeba histolytica surfacelectins.

In another preferred embodiment, the αGal oligosaccharide, orpharmaceutically acceptable derivative thereof, is coupled to a ligandthat binds targets located on parasites and may be used in the treatmentof parasitic diseases. Ligands that target pathogenic organisms mayinclude, but are not limited to, any oligosaccharide motif used bypathogens to recognize host cells which has been described or may beroutinely determined through techniques known in the art. Sucholigosaccharides have been shown to be effective anti-adhesive agentsagainst pathogens including but not limited to Bordetella pertussis,Citrobacter freundii, Escherichia coli, Haemophilus influenzae,Helicobacter pylori, Mycoplasma pneumoniae, Proteus mirabilis,Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescens,Shigella flexneri, Staphylococcus saprophyticus, Streptococcus mutans,Streptococcus pneumoniae, Streptococcus sanguis, Vibrio cholerae,Cryptosporidium parvum, and Entiamoeba histolytica (See generally Zopfet al., 1996, The Lancet, 347:1017-1021, which is incorporated byreference in its entirety).

In another preferred embodiment, the αGal oligosaccharide, orpharmaceutically acceptable derivative thereof is conjugated to the CD22ligand NeuAcα2-6Galβ1-4GlcNAc. This conjugate may be used to deliverαGal and thus anti-αGal-mediated complement lytic attack to cellsexpressing the CD22 molecule which is required for B-T-cell cooperationduring lymphocyte activation to regulate immunosuppression.

The αGal oligosaccharide component of the pharmaceutical composition maybe administered alone or in combination with other agents useful inattenuating xenograft rejection, including conventional nonspecificimmunosuppressive agents, including but not limited to, steroids,cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone,prednisone, azathioprine, FK-506, 15-deoxyspergualin, and otherimmunosuppressive agents known in the art. In a further embodiment, thepharmaceutical compositions comprise an antibiotic agent selected fromthe group consisting of tetracycline, metronidazole, amoxicillin, βlactamases, aminoglycosides, macrolides, quinolones, fluoroquinolones,cephalosporins, erythromycin, ciprofloxacin, and streptomycin. In anadditional embodiment, the pharmaceutical composition comprises ananti-inflammatory. Such anti-inflammatories include, but are not limitedto, glucocorticoids and the nonsteroidal anti-inflammatories,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, ε-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

5.1.1. αGAL OLIGOSACCHARIDE COMPOSITION PRODUCTION

While, in theory the αGal oligosaccharides of the invention can bepurified from biological tissue or cell culture, or produced usingclassical organic chemistry synthetic techniques known in the art, suchderivation of αGal oligosaccharide in the quantity needed according tothe methods of the invention, is impractical. Accordingly, it ispreferred that the oligosaccharides of the present invention areprepared using enzymatic processes.

Donor saccharide moieties and acceptor moieties for enzymatic synthesisof αGal oligosaccharides may be commercially available and/or may beobtained through organic synthesis applying techniques known in the art.Activated saccharides generally consist of uridine or guanosinediphosphate and cytidine monophosphate derivatives of the saccharides inwhich the nucleoside mono- and diphosphate serves as a leaving group.Thus, the activated saccharide may be a saccharide-UDP, asaccharide-GDP, or a saccharide-CMP. Nucleoside monophosphates arecommercially available, may be prepared from known sources such asdigested yeast RNA (see e.g., Leucks et al,. 1979, J. Am. Soc.101:5829), or routinely prepared using known chemical synthetictechniques (see e.g, Heidlas et al., 1992, Acc, Chem. Res. 25:307;Kochetkov et al., 1973, Adv. Carbohydr. Chem. Biochem. 28:307). Thesenucleoside monophosphates may then be routinely transformed intonucleoside diphosphates by kinase treatment. For review, see Wong etal., 1994, Enzymes in Synthetic Organic Chemistry, Pergamon Press,Volume 12, pp 256-264.

Glycosyltransferase enzymes for synthesizing the compositions of theinvention can be obtained commercially or may be derived from biologicalfluids, tissue or cell cultures. Such biological sources include, butare not limited to, pig serum and bovine milk. Glycosyltransferases thatcatalyze specific glycosidic linkages may routinely be isolated andprepared as described in International Patent Publication No. WO93/13198 (published Jul. 8, 1993), which is herein incorporated byreference in its entirety. Alternatively, the glycosyltransferases canbe produced through recombinant or synthetic techniques known in the art(For review, see Wong et al., 1994, Enzymes in Synthetic OrganicChemistry, Pergamon Press, Volume 12, pp 275-279).

The compositions of the invention are preferably synthesized usingenzymatic processes (see e.g., U.S. Pat. No. 5,189,674, andInternational Patent Publication No. 91/16449, published Oct. 31, 1991,each of these references is herein incorporated by reference in itsentirety). Briefly, a glycosyltransferase is contacted with anappropriate activated saccharide and an appropriate acceptor moleculeunder conditions effective to transfer and covalently bond thesaccharide to the acceptor molecule. Conditions of time, temperature,and pH appropriate and optimal for a particular saccharide unit transfercan be determined through routine testing; generally, physiologicalconditions will be acceptable. Certain co-reagents may also bedesirable; for example, it may be more effective to contact theglycosyltransferase with the activated sugar and the acceptor moleculein the presence of a divalent cation. Optionally, an apparatus asdescribed by U.S. Pat. No. 5,288,637, is used to prepare suchcompositions (this reference is herein incorporated by reference in itsentirety).

By way of example, the αGal trisaccharide (Galα1-3Galβ1-4GlcNAc) may besynthesized by contacting N-acetylglucosamine with UDP-galactose and aβ-N-acetylglucosaminoside β1-4 galactosyltransferase. The productdisaccharide is contacted with UDP-galactose and a β-galactoside β1-3galactosyltransferase, and techniques known in the art are applied toconcentrate the resulting trisaccharide (See, for example, Section 6.1).

In another example, the αGal pentasaccharide(Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc) is synthesized by contactinglactose (galactoseβ1-4glucose) with UDP-N-acetylglucosamine and agalactoside β1-3 N-acetylglucosaminyl transferase. The producttrisaccharide is contacted with UDP-Gal and a β-N-acetylglucosaminosideβ1-4 galactosyltransferase and the resulting tetrasaccharide iscontacted with UDP-galactose and a β-galactoside β1-3galactosyltransferase. The resulting pentasaccharide is concentratedusing techniques known in the art (See, for example, Section 6.2).

While glycosyltransferases are highly stereospecific andsubstrate-specific, minor chemical modifications are tolerated on boththe donor and acceptor components. Accordingly, the oligosaccharidecomponents of the invention may be synthesized using acceptor and/ordonor components that have been modified so as not to interfere withenzymatic formation of the desired glycosidic linkage. The ability ofsuch a modification not to interfere with the desired glycosidic linkagemay routinely be determined using techniques and bioassays known in theart, such as, for example, labelling the carbohydrate moiety of theactivated sugar donor, contacting the acceptor and donor moieties withthe glycosyltransferase specific for forming the glycosidic linkagebetween the donor and acceptor moieties, and determining whether thelabel is incorporated into the molecule containing the acceptor moiety.

Examples of modified αGal oligosaccharides (i.e., derivatives)encompassed by the invention include, but are not limited to, salts andsulfate substitutes of αGal oligosaccharides, as well as αGaloligosaccharides in which one or more or the pyran rings has beensubstituted with a piperidine ring system and/or a tetrahydrothiopyranring system.

αGal oligosaccharide aza sugars in which the oxygen of one or more ofthe pyran rings of the oligosaccharide is replaced with nitrogen to forma piperidine ring system may be prepared by enzymatic methods known inthe art using the appropriate aza saccharide as the acceptor substrate.Alternatively, aza sugar donor moieties may be transferred by thecorresponding glycosyltransferase for the natural sugar. Aza glucose canbe isolated from natural sources and converted to the aza lactose by theaction of a galactosyltransferase in the presence of a galactose donorsuch as for example, UDP-galactose.

The pharmaceutical composition of the invention may also comprise αGaloligosaccharide thio sugars, in which the oxygen of one or more of thepyran rings of the oligosaccharide is replaced with sulfur to form atetrahydrothiopyran ring system. The monothiosaccharide may be preparedby known organic chemical techniques from the correspondingmonosaccharide and the αGal oligosaccharide derivative thio sugar may beprepared applying enzymatic methods and using the appropriate thiosaccharide as the acceptor substrate.

The αGal oligosaccharide or pharmaceutically acceptable derivativethereof, is optionally associated with or conjugated to other molecules,including but not limited to biologically inert molecules, proteins(e.g., monoclonal antibodies), glycoproteins, lipids, glycolipids, andcarbohydrates. This association or conjugation may be the result of adirect interaction between the reducing end of the αGal oligosaccharideand the other molecule through an ionic or chemical bond. Alternatively,a linking group may mediate this association or conjugation.

The chemistry necessary to link the reducing end of the αGaloligosaccharide with the other molecule or with the linking groupintermediary and to link the αGal oligosaccharide-linking group complexto the other molecule is well known in the field of linking chemistry.For example, a bond between the reducing end saccharide and a linkinggroup can be formed by reacting an aldehyde or carboxylic acid at C₁ ofthe reducing end saccharide or any aldehyde or carboxylic acid groupintroduced onto the reducing end saccharide by oxidation, with thelinking group, to form a suitable bond such as --NH--, --N(R') where R'is C₁₋₂₀ alkyl, a hydroxyalkylamine, an amide, an ester, a thioester, athioamide.

Additionally, the bond between the reducing end saccharide and thelinking group can be formed by reacting the C₁ hydroxyl group, in thepyranose form, with an acylating agent and a molecular halide, followedby reaction with a nucleophile to form a suitable bond such as NH--,--N(R')-- where R' is C₁₋₂₀ alkyl, --S-- and --O--. This type of linkingchemistry is further described by Stowell et al, 1980, Advances inCarbohydrate Chemistry and Biochemistry, 37: 225-281.

The oligosaccharide portion can be bound directly to the other molecule(e.g., multivalent support) via the free anomeric carbon of the reducingend saccharide. Alternatively, the reducing end saccharide can be boundvia a phenethylamine-isothiocyanate derivative as described by Smith etal., (1978, Complex Carbohydrates part C, Methods in Enzymology, Ed byV. Ginsburg, Volume 50, pp 169-171 or a glycine amine derivative asdescribed in Section 6.4.).

In a specific embodiment, αGal oligosaccharides associated or conjugatedwith monoclonal antibodies, or fragments thereof, targetcomplement-mediated lytic attack to tissue or cell types expressing theantigen recognized by the monoclonal antibody. Techniques foridentifying monoclonal antibodies which recognize a specificcell-surface antigen are known in the art. See generally, Harlow, E.,1988, Antibodies a Laboratory Manual, Cold Spring Harbor, Ed. by Harlowand Lane.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to produce single chainantibodies against a distinguishing antigen of interest. Single chainantibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single chainpolypeptide.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab')₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

5.2. ASSAYS FOR COMPETITIVE INHIBITION OF ANTI-αGAL ANTIBODY BINDING BYαGAL OLIGOSACCHARIDE COMPOSITIONS

The invention is based in part on the discovery that administration ofoligosaccharides containing the αGal motif in sufficient quantities tobind to and neutralize anti-αGal antibodies is sufficient to attenuatexenograft rejection of pig to old world monkey/human xenografts in vivo.Thus, the ability of compositions comprising αGal oligosaccharides orpharmaceutically acceptable derivatives thereof to remove, bind, and/orneutralize anti-αGal antibodies is indicative of the ability of thecomposition to attenuate xenograft rejection.

Quantification of circulating anti-αGal antibodies in the serum of anindividual and the ability of compositions comprising αGaloligosaccharides or pharmaceutically acceptable derivatives thereof toremove, bind, and/or neutralize anti-αGal antibodies can be routinelydetermined applying the immunoassays described in Sections 6.3, 6.4, 6.5as well as other immunoassays known in the art which may be routinelyadapted for such determination. Such immunoassays, include, but are notlimited to, competitive and non-competitive assay systems usingtechniques such as western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), "sandwich" immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays(e.g., hemagglutination), complement-fixation assays, immunoradiometricassays, fluorescent immunoassays and protein A immunoassays. Theseassays may further be applied to determine the dosage of the αGaloligosaccharide composition of the invention to be administered (seeSection 5.6.2) and to monitor the neutralization and/or removal ofanti-αGal antibodies by the αGal oligosaccharide compositions of theinvention.

Immunoassays known in the art may also be routinely odified to determinethe affinity of cell surface ligand components of the compositions ofthe invention (e.g., monoclonal antibodies) to bind to theircell-surface bonding partners.

While assays for measuring circulating anti-αGal antibodies can beaccomplished by various immunological methods, ELISA assays have theadvantage in that they can be standardized for the immobilized ligand,are reproducibly quantitative, and can be scored for differentimmunoglobulin isotypes by the use of appropriate secondary detectionreagents. ELISA capture ligands for testing human or old world monkeyserum antibodies can employ fixed cells, glycolipids, glycoproteins, oroligosaccharides. Such capture ligands include but are not limited tomouse laminin (see Section 6.3), PK-15 pig kidney cells (see Section6.5), immobilized BSA-αGal neoglycoconjugates, and immobilized αGaloligosaccharides (see Section 6.4). Covalent immobilization of aspecific αGal oligosaccharide(s) onto the ELISA immobilized surface,using techniques known in the art, permits the predetermined depositionof known antigenic ligands by covalent chemistry. In addition todetermining the ability of αGal oligosaccharides or pharmaceuticallyacceptable derivatives thereof to bind to and/or neutralize anti-αGalantibodies, such an ELISA is also useful in clinical monitoring ofanti-αGal antibodies in blood of patients in advance of, and followingxenotransplantation of organs from animals that express the αGalepitope.

Generally, an ELISA assay may comprise contacting serum isolated from apatient, using techniques known in the art, with anti-αGal antibodyligand, in the presence or absence of an αGal oligosaccharide orpharmaceutically acceptable derivative of the invention, under constantconditions. After washing away excess serum, the extent of anti-αGalantibody binding to the ligand is assessed using techniques known in theart, including, but not limited to, adding a secondary antibody with areporter group (e.g., goat anti-human IgG-alkaline phosphatase),followed by a suitable read-out reagent (e.g., alkaline phosphatasesubstrate p-nitrophenyl phosphate, or pNPP). Reduced levels of anti-αGalantibody capture by the ligand in serum treated with αGaloligosaccharides of the invention or pharmaceutically acceptablederivatives thereof relative to control samples extracted from thepatient prior to or in the absence of treatment with the αGaloligosaccharide or pharmaceutically acceptable derivative of theinvention, indicates that the αGal oligosaccharide or pharmaceuticallyacceptable derivative thereof competitively inhibits binding ofanti-αGal antibodies to the αGal motif containing ligand.

In a specific embodiment, αGal oligosaccharide glycine amide derivativesare synthesized by linking glycine to the reducing monosaccharide. Theglycine derivative is then applied, under.alkaline conditions, tomicrotiter plate wells that have been derivatized with N-oxysuccinimidegroups. The reduced amino groups displace the succinimide group forminga stable covalent bond. The plate is then blocked with a solutioncontaining non-glycosylated protein (e.g., albumin) to reducenon-specific binding to plastic and test sera or plasmas in the presenceor absence of an unbound α-Gal oligosaccharide or pharmaceuticallyacceptable derivative of the invention, are added to the plate andincubated for an interval to permit attachment of antibodies to thebound oligosaccharide derivatives. After washing the plate repeatedlywith blocking solution (containing albumin and detergent), a secondaryantibody with a reporter group (e.g., goat anti-human IgG-alkalinephosphatase) is added and allowed to bind the anti-αGal immunoglobulinsadsorbed onto the immobilized oligosaccharide. After washing the plateagain, a suitable read-out reagent (e.g., alkaline phosphatase substratep-nitrophenyl phosphate, or PNPP) is then added and color development ismeasured using a microtiter plate reader.

The addition of serial dilutions of sera in the ELISA assays permits thedetermination of reactive antibody titers, which can serve to quantitatethe circulating anti-αGal activity. In sera drawn from patients treatedwith soluble oligosaccharide only the unblocked immunoglobulin is freeto bind the immobilized ligand (with the caveat that competition for theantigen-binding site of the antibody can result in the displacement ofsoluble oligosaccharide by immobilized ligand displaying greateraffinity or activity). Specific immunoglobulin isotypes can be monitoredby using appropriate reagents, e.g., anti-human IgG or IgM.

In another embodiment, quantification of anti-αGal antibodies and/or theextent of xenoreactive antibody neutralization or removal upon treatmentwith the αGal oligosaccharides of the invention is measured using a cellcytotoxicity assay such as that described in Section 6.5. Such an assaymay comprise contacting serum, isolated from a patient using techniquesknown in the art, with αGal expressing cell monolayers (e.g., pig kidneycells (PK-15), pig aortic endothelial cells, and mouse aorticendothelial cells (MAE). Preferably, these cells are from the samespecies as the donor and most preferably from the same tissue-type asthe tissue to be transplanted), in the presence or absence of an αGaloligosaccharide or pharmaceutically acceptable derivative of theinvention, under constant conditions, such as, for example, 1 hour at37° C. According to this assay, cytotoxicity is mediated by eitherendogenous complement, or the sera are heat inactivated (e.g., throughheating the sera at 56° C. for 30 minutes) and exogenous complement(e.g., from rabbit or guinea pig) is added. After washing away excessserum, the cells are treated with viable dye mix "live-dead" (calceinAM/ethidium homodimer) which is commercially available in the form of aLive/Dead cytotoxicity kit (Molecular Probes Inc., Eugene, Oreg.) andscored for viability using fluorescence microscopy. Staining with the"live/dead" dye mix allows for clear distinction between live cells,which show cytoplasmic green fluorescence and dead cells, which showdark cytoplasm and red fluorescent nuclei. The extent of cell lysis thatis complement-mediated may be determined by comparing the resultsobserved in the control for which there has been no inactivation ofcomplement, with the lysis observed with complement that has beeninactivated through heat treatment.

The invention also encompasses animal-based model systems, which mayinclude baboon, other old world monkeys, or other animals having serumthat contains anti-αGal antibodies. Such animal models may assess theability of compositions containing an αGal oligosaccharide orpharmaceutically acceptable derivative as an active ingredient to bindto and/or neutralize anti-αGal antibodies, attenuate the rejection of axenotransplant expressing the αGal epitope and/or to suppress Blymphocytes expressing anti-αGal idiotypes. Generally, this assay mayinvolve exposing animal models to a compound containing an αGaloligosaccharide or pharmaceutically acceptable derivative as an activeingredient, at a sufficient concentration and for a time sufficient toelicit the desired effect in the exposed animals. The response of theanimals to the exposure may be monitored by assessing the level ofanti-αGal reactive antibodies in the serum of the animal, evaluating theappearance of the xenografted organ, and/or quantitating B lymphocytesexpressing anti-αGal idiotypes. Dosages of test agents may be determinedby deriving dose-response curves, as discussed in Section 5.6.1, below.

The assays described herein may be applied to routinely determine whichαGal oligosaccharides, or pharmaceutically acceptable derivativesthereof, are able to bind and thereby neutralize and/or remove anti-αGalantibodies and the optimal concentrations for doing so. The assays mayalso be applied to determine the relative binding affinity for anti-αGaldemonstrated by the αGal oligosaccharide compositions of the invention.Once αGal oligosaccharides, or pharmaceutically acceptable derivativesdisplaying the greatest binding affinity have been identified, thesecompounds are optionally combined and the assays are routinely appliedto determine optimal combination concentrations for the pharmaceuticalcompositions of the invention.

Applying these assays, the relative anti-αGal binding activity that aαGal oligosaccharide or pharmaceutically acceptable derivative exhibitsagainst the anti-αGal antibody profile of the serum of an individual maybe determined and the αGal oligosaccharide and/or pharmaceuticallyacceptable derivative combination formulation best suited forneutralizing and/or binding the anti-αGal profile of an individual canbe determined (see Section 5.3).

The αGal oligosaccharide or pharmaceutically acceptable derivativethereof may then be combined with suitable pharmaceutically acceptablecarriers and administered by techniques known in the art, such as thosedescribed in Section 5.6 infra.

Other methods for assaying the extent of xenoreactive antibodyneutralization and/or removal will be known to the skilled artisan andare within the scope of the invention.

5.3. FORMULATION OF PATIENT SPECIFIC PHARMACEUTICAL COMPOSITIONS

The human body produces anti-αGal antibodies in response to commonbacterial antigens present in gastrointestinal and respiratory systems(Galili et al., 1988, Infection and Immunity, 56(7):1730-1737). Thevariability of intestinal and respiratory bacterial flora, as well asthe diversity of immune response among individuals, suggests theexistence of subpopulations and variable profiles of anti-αGalantibodies in potential recipients.

In particular embodiments, the invention provides methods forformulating a pharmaceutical composition which comprises αGaloligosaccharides and/or pharmaceutically acceptable derivatives thereofthat are able to competitively inhibit binding of the anti-αGal antibodyprofile of an individual. Such methods are achieved by withdrawing serumfrom the individual using techniques known in the art and testing theability of a panel of αGal oligosaccharides, or pharmaceuticallyacceptable derivatives thereof, to determine which particular αGaloligosaccharides or derivatives competitively inhibit anti-αGal bindingto donor endothelium (or cell lines such as PK-15 pig kidney cells, pigaortic endothelial cells, or MAE mouse aortic endothelial cells).Preferably, these cells are from the same species as the donor and mostpreferably from the same tissue-type as the tissue to be transplanted).The αGal oligosaccharides thereby identified as having the highestactivity in inhibiting the binding of anti-αGal antibodies in thepatient's serum are then used as components of an ex vivo depletiondevice or as a pharmaceutical composition comprising them to treat thepatient.

The ability to formulate a therapeutic composition to contain only αGaloligosaccharides or pharmaceutically acceptable derivatives thereof,demonstrating high activity in neutralizing the anti-αGal antibodyprofile of a patient minimizes the dosage of antibody-neutralizingoligosaccharide to be delivered. This ability to formulate a therapeuticcomposition to contain only those αGal oligosaccharides orpharmaceutically acceptable derivatives thereof demonstrating highactivity in inhibiting binding of anti-αGal antibodies to donorendothelium is extremely valuable since the risk of side effectsincrease with the concentration of the αGal oligosaccharide orpharmaceutically acceptable derivative thereof.

Tissue sources and cell lines expressing cell-surface antigensrecognized by anti-αGal antibodies are readily available. For examplepig kidney cells (PK-15, ATCC CCL 33, Rockville, Md.) and pig aorticendothelial cells (AG 08472, N.I.A. Aging Cell Culture Repository,Camden, N.J.) are available from cell culture repositories. In oneembodiment, potential recipient serum is tested in assays, such as thosedescribed infra, for the ability to bind to and optionally to kill cellsexpressing cell-surface antigens recognized by anti-αGal antibodies inthe presence of αGal oligosaccharides or pharmaceutically acceptablederivatives thereof. αGal oligosaccharides and pharmaceuticallyacceptable derivatives thereof found to be effective in inhibitingbinding of the anti-αGal antibodies to the cells are preferably thentested over a range of concentrations using techniques known in the art.In a specific embodiment of the invention, the pharmaceuticalcomposition of the invention comprises a plurality of αGaloligosaccharides and pharmaceutically acceptable derivatives thereofdetermined to be effective in neutralizing binding of the recipient'santi-αGal antibody profile to cells expressing cell surface antigensrecognized by anti-αGal antibodies. In particular embodiments, thepharmaceutical composition comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10distinct αGal oligosaccharides or pharmaceutically acceptablederivatives thereof determined to have activity in neutralizing theanti-αGal antibody profile of the recipient.

The invention therefore provides methods by which to identify αGaloligosaccharides and pharmaceutically acceptable derivatives thereofthat neutralize the anti-αGal antibody profile of a potential recipient,and by which pharmaceutical compositions containing these αGaloligosaccharides and pharmaceutically acceptable derivatives alone or incombination are routinely formulated. The invention further providesmethods for treating or preventing hyperacute rejection of xenografts inhumans and old world monkeys, by administering the pharmaceuticalcompositions of the invention.

The invention thus provides methods for formulating on apatient-to-patient basis, a pharmaceutical composition comprising αGaloligosaccharides or pharmaceutically acceptable derivatives thereof thatare known to be effective in neutralizing the anti-αGal antibody profilein an individual. Accordingly, the αGal oligosaccharides of theinvention may be administered alone or in combinations for effectivelyneutralizing different anti-αGal antibody profiles. The in vitro assaysdescribed in Sections 5.2, 6.3, 6.4 and 6.5 may be applied to assess theability of the α-Gal oligosaccharide compositions of the invention tobind the anti-αGal profiles of different potential recipients.

5.4. DIAGNOSTIC USES OF THE αGAL OLIGOSACCHARIDE COMPOSITIONS OF THEINVENTION

The present invention is based in part on the discovery that the abilityto neutralize anti-αGal antibody directed complement-mediated lyticattack is determinative of whether a xenograft will be accepted.Accordingly, concentrations of these antibodies and/or B-lymphocytesbearing anti-αGal idiotypes are likely to approximate the severity ofxenograft rejection.

The concentration of anti-αGal antibodies in the serum of a potentialrecipient is likely to have a direct correlation with the severity inwhich the recipient will reject a xenograft, i.e., the higher theconcentration of these antibodies in the serum of an individual, themore severe the rejection of a xenograft. Quantification of anti-αGalantibody concentrations in the serum of a potential recipient may alsoaid in designing an effective therapy regimen for the patient. Assaysdescribed infra (Sections 5.2, 6.3, 6.4 and 6.5) and known in the artmay be applied to determine the concentration of anti-αGal antibodies inthe serum of a potential recipient.

The number of lymphocytes bearing anti-αGal idiotypes in a potentialrecipient may also be used as a predictor of the severity of xenograftrejection. A high number of lymphocytes bearing anti-αGal idiotypes islikely to be associated with a more severe rejection of a xenograft, aswell as faster regeneration of anti-αGal antibodies. Methods forquantifying lymphocytes bearing anti-αGal idiotypes in a potentialrecipient are known to those in the art. B-lymphocytes including thosebearing anti-α-Gal idiotypes may be isolated from lymph nodes (via lymphnode aspirate or biopsy) or peripheral blood using techniques known inthe art, such as, for example, (+) or (-) selection by immunomagneticbeads (Dynal, A. S. Norway).

One method of quantifying B-lymphocytes bearing anti-αGal idiotypesinvolves isolating mononuclear cells from patients' peripheral blood bythe histopaque method (Sigma, St. Louis, Mo.). According to this method,the cells are incubated with an αGal oligosaccharide composition coupledwith fluorescein isothiocyanate using techniques known in the art inorder to visualize cells with the surface-expressed idiotype. Inaddition to the FITC-labeled αGal oligosaccharide composition (greenfluorescence), the cells are also stained with PerCP-labeled (redfluorescence) B-lymphocyte-specific monoclonal antibody Anti-Leu 12(Becton Dickinson, San Jose, Calif.). This double staining procedure isfollowed by fluorescence analysis with Becton/Dickinson's FACScan usingthe two-color program. The subsets of B-lymphocytes bearing specificanti-αGal idiotypes can then be accurately counted.

5.5. THERAPEUTIC USES OF THE αGAL OLIGOSACCHARIDE COMPOSITIONS OF THEINVENTION

The presence of xenoantibodies comprises the principal, and mostdevastating, problem in attempts to xenotransplant animal organs intohumans and old world monkeys. For example, pig hearts transplanted intobaboons or cynomologous monkeys turn dark and necrotic in as little as5-10 minutes following vascular connection, a phenomenon known ashyperacute rejection (HAR). It has been estimated that a 1-2 weekregimen of xenoantibody neutralization or removal would overcome thehyperacute rejection barrier. Evidence to support this concept arisesfrom experience with ABO-mismatched organ transplants (Cooper et al.,1993, Transplant. 56:769-777; Alexandre et al., 1987, Transplant Proc.19:4538; and Bennett et al., 1987, Transplant Proc. 19:4543). Accordingto this concept, after the hyperacute rejection barrier has beenovercome, an "accommodation" takes place and the organ can be stabilizedwith routine immunosuppression regimens directed to suppressing cellularrejection (Bach et al., 1991, Transplant. Proc. 23:205; Simpson et al.,1989, Xenograft, Elsevier, N.Y., 25:273-284; Michler, 1987,Transplantation 44(5)632-636).

The majority of xenoantibodies present in the blood of humans and oldworld monkeys recognize an antigenic epitope on donor vascularepithelium containing a αGal motif (Galα1-3Gal). The present inventionis based in part on the discovery that binding of anti-αGal antibodiesto donor endothelium and the complement-mediated lytic attack directedthereby, are the determining factors leading to hyperacute rejection ofxenografts and that removal or neutralization of anti-αGal antibodiesattenuates xenograft rejection. Accordingly, the invention relates tomethods which utilize αGal oligosaccharide compositions to interferewith (i.e., competitively inhibit) the ability of anti-αGal antibodiesto bind to donor organ endothelium. According to the invention, αGalantibody neutralization or depletion may be achieved through either orboth in vivo and ex vivo treatment of host serum or serum to beadministered to the host.

In one embodiment, anti-αGal antibodies are depleted by passing blood tobe administered to a patient over αGal oligosaccharides orpharmaceutically acceptable derivatives of the invention that have beenbound either directly or through a linker, to a biocompatible solidsupport using methods known in the art, including, but not limited tothose techniques described in Sections 5.1.1 and 6.4. According to thisembodiment, blood of the patient is passed ex vivo over the αGal-supportmatrix complex and then transfused into the patient using techniquesknown in the art. Extracoporeal reactors, such as dialysis orplasmapheresis machines, are readily adapted for this procedure bymethods known in the art.

A preferred method of neutralizing anti-αGal antibodies involves theintravenous administration of αGal oligosaccharide(s) orpharmaceutically acceptable derivative(s) thereof in sufficient quantityto block binding of the circulating antibody to the donor endotheliumand thereby prevent anti-αGal antibody directed complement-mediatedlytic attack of the transplanted tissue. Methods and compositions forformulating and administering the pharmaceutical compositions of theinvention are known in the art, and include but are not limited to thatdescribed in Sections 5.2, 5.3 and 5.6 infra.

Assays which can be used to determine whether administration of aspecific composition attenuates xenograft rejection are discussed infra(see, Sections 5.2, 6.3, 6.4 and 6.5). These assays can indicate whichαGal oligosaccharide or pharmaceutically acceptable derivative thereofhas the desired therapeutic efficacy in attenuating xenograft rejectionand additionally may be applied to assay for the ability of combinationsof αGal oligosaccharides and/or pharmaceutically acceptable derivativesto competitively inhibit binding of anti-αGal antibodies to theendothelium of the donor organ.

The αGal oligosaccharide compositions of the invention may beadministered alone or in combination with other therapeutic agents,including but not limited to, antibiotics, steroidal and non-steroidalanti-inflammatories, and conventional immunotherapeutic agents.Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the αGal oligosaccharide compositionsof the invention include, but are not limited to, steroids,cyclosporine, cyclosporine analogs, methylprednisolone, and azathioprineFK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells. Combinations may beadministered either concomitantly, e.g., as an admixture, separately butsimultaneously or concurrently; or sequentially. This includespresentations in which the combined agents are administered together asa therapeutic mixture, and also procedures in which the combined agentsare administered separately but simultaneously, e.g., as throughseparate intravenous lines into the same individual. Administration "incombination" further includes the separate administration of one of theagents given first, followed by the second. The invention alsoencompasses the use of a combination of distinct αGal oligosaccharides,e.g., αGal trisaccharide in combination with αGal pentasaccharide.

In a specific embodiment, the therapeutic method of the invention iscarried out as monotherapy, i.e., as the only agent provided forattenuating xenograft rejection. In preferred embodiments, this therapyinvolves delivery of αGal trisaccharide and/or αGal pentasaccharide.

It is a primary object of this invention to provide a method andassociated compositions for attenuating xenograft rejection or toalleviate trauma caused by anti-αGal antibody directed complementactivation by interfering with anti-αGal antibody binding to cellsurfaces, in particular donor organ endothelium. Accordingly, thepharmaceutical compositions of the invention may be administered alone,together with, or in seriatim with other therapy regimens for reducingthe extent of binding of anti-αGal antibody to donor organ cells ortissue. In one embodiment, administration of the αGal oligosaccharide ofthe invention is combined with parenteral administration and/orextracorporeal treatment with column immobilized anti-agal idiotypicantibodies (for example, see U.S. Pat. No. 5,560,911). Other regimensthat may accompany neutralization and/or depletion of anti-αGalantibodies using αGal oligosaccharides or pharmaceutically acceptablederivatives, include but are not limited to, extracorporeal treatmentwith column immobilized human anti-animal idiotypic antibodies (forexample, see U.S. Pat. No. 5,560,911), plasmapheresis (in which allantibodies or specifically, one or more antibody types specific forantigenic epitopes on the surface of donor endothelial cells have beenremoved from the plasma) and perfusion of blood to be administered tothe patient through organs, tissue or cells expressing αGal antigens(such as, for example, hearts, kidneys, erythrocytes, and cell linesderived from pig kidney, pig aortic endothelium, mouse endothelium,etc.).

Depletion of circulating anti-αGal antibody is a temporary solution,which can overcome the HAR crisis. But the antibody-producing Blymphocytes continue to produce antibody, which can pose a danger oflonger-term antibody-mediated vascular rejection. These B lymphocytesbear on their membrane surface immunoglobulin with the αGal-bindingdomain exposed (Geller et al. 1993, Transplantation 55:168-172).

In particular embodiments, the administration of αGal oligosaccharidesor pharmaceutically acceptable derivatives thereof targets cytocidalagents or complement-mediated lytic attack to B lymphocytes expressinganti-αGal idiotypes, thereby ablating the host's ability to mount theantibody response. This administration may be prior to, during, orsubsequent to xenotransplantation and is directed toward reducing boththe production and regeneration of anti-αGal antibodies. The dosage andfrequency of administration is determined by the number ofidiotype-bearing B lymphocytes present in peripheral blood, which may bequantified using techniques described in Section 5.4 or known in theart.

One embodiment encompasses the administration of an αGal oligosaccharidechemically linked to a cytocidal agent. Such cytocidal agents include,but are not limited to, toxins and cytotoxic drugs. Effective targetingof the αGal oligosaccharide/cytocidal agent complex to B lymphocytesbearing anti-αGal idiotypes requires avoidance of circulating anti-αGalantibodies to permit access to the B-cells. Accordingly, it is preferredthat this complex is administered following ex vivo depletion ofanti-αGal antibodies. Such depletion treatment may include, but is notlimited to, extracorporeal exposure of host serum or plasma to αGaloligosaccharides, derivatives and/or anti-αGal idiotypic antibodiesbound to a biologically inert matrix or a pig organ.

In another embodiment, αGal oligosaccharide compositions of theinvention are used to target anti-αGal antibodies to the B-cells thatproduced them via drawing in a complement-mediated lytic attack.According to this embodiment, a complex comprising at least two αGalepitopes is administered to a patient, preferably following ex vivotreatment to deplete anti-αGal antibodies in the serum of the patient.While not wishing to be bound by theory, it is proposed that one of theαGal epitopes of this complex would be bound by the B-cell's surfaceimmunoglobulin, while the remaining αGal epitope(s) would be displayedoutward, serving as surface ligands for the circulating anti-αGalantibodies. In specific embodiments, the complex comprises at least 2,3, 4, 5, 6, 7, 8, 9, 10, 15 or 20, αGal epitopes as a single molecule ormultivalent. In preferred embodiments, the complex comprises 2 or 3 αGalepitopes. In another preferred embodiment, all but one, of the αGalepitopes of the complex are "masked" by a labile group as described inSection 5.1.1. While not wishing to be bound by theory, it is believedthat by masking all but one αGal epitope of the complex, this treatmentwould minimize the risk of the formation of large circulating immunecomplexes, which could give rise to congestive events in the renal,glomerular, hepatic or pulmonary capillary networks, etc. According tothis embodiment, the exposed αGal epitope binds the B-cell surfaceimmunoglobulin and the masking group is gradually removed by natural orpharmacological intervention, exposing the previously masked αGal group,and bringing about complement-mediated lytic attack of the B-cells.Effective targeting of the αGal oligosaccharide/cytocidal agent complexto B lymphocytes bearing anti-αGal idiotypes requires avoidance ofcirculating anti-αGal antibodies to permit access to the B-cells.Accordingly, it is preferred that this complex is administered while theanti-αGal antibody titer has been reduced to negligible by treatmentsthat include, but are not limited to: ex vivo depletion using a columnbearing an αGal-oligosaccharide or anti-αGal idiotypic antibodies, or byusing an organ from the donor species; in vivo infusion of soluble αGaloligosaccharide; absorption of anti-αGal antibodies by the xenograft; ora combination of these events.

In another embodiment, the ex vivo depletion of anti-αGal antibodiesusing αGal oligosaccharides or pharmaceutically acceptable derivativesof the invention is followed by the administration of anti-αGalidiotypic antibodies capable of binding to the surface of B lymphocytesand mediating the destruction of these cells either directly (e.g.,where the antibodies are associated with or conjugated to a cytocidalagent), or through triggering complement-mediated lytic attack ifconjugated to or associated with a cytocidal agent. Cytotoxicrecombinant ScFvs may be generated using techniques known in the art.See, e.g., George, A. J. T., The Second Annual IBC InternationalConference on Antibody Engineering, San Diego, Calif., Dec. 16-18, 1991,incorporated herein by reference in its entirety.

The invention also encompasses the administration of αGaloligosaccharides of the invention to target anti-αGal antibody directedcomplement-mediated lytic attack to specific tissue and cell types.Thus, the invention provides for targeting complement-mediated lyticattack to a tissue, cell type, or organism expressing a distinguishingmarker, by administering an effective amount of a αGal oligosaccharideor pharmaceutically acceptable derivative of the invention comprising aligand for such marker. Examples of such arkers include, but are notlimited to, tumor specific ntigens to which antibodies have beendeveloped or may be routinely identified and generated using techniquesknown in the art.

In one embodiment, an αGal oligosaccharide composition of the inventioncomprising an autoantigenic peptide is administered to target anti-αGalantibodies to the MHC of autoreactive lymphocytes. In specificembodiments, the administered composition comprises one or more peptidesselected from the group consisting of myelin basic protein peptides inMultiple Sclerosis, pancreatic islet autoantigenic peptides on p54 injuvenile (type I or autoimmune) diabetes mellitus, acetylcholinereceptor peptides in myasthenia gravis and collagen peptides inrheumatoid arthritis).

In another embodiment, an αGal oligosaccharide composition of theinvention comprising a ligand for a molecule required for lymphocyteactivation is administered to target anti-αGal antibody mediated lyticattack to cells expressing this molecule. In a specific embodiment, theadministered composition comprises an αGal oligosaccharide and the CD22ligand NeuAcα2-6Galβ1-4GlcNAc. In another specific embodiment, theadministered composition comprises a monoclonal antibody that bindsCD22. Such monoclonal antibodies may be routinely generated usingtechniques known in the art or obtained commercially. This treatment isdirected toward complement-mediated lytic attack of cells expressing theCD22 molecule required for B-T-cell cooperation during lymphocyteactivation.

In another embodiment, an αGal oligosaccharide composition of theinvention comprising a ligand bound by a pathogenic virus or by avirally infected cell is administered to target anti-αGalantibody-mediated lytic attack to the virus or infected cell. Inspecific embodiments, the administered composition comprises CD4-derivedpeptide bound by gp120 of HIV (from the D1 domain of CD4 and distinctfrom the MHC-binding region) and/or chemokine receptor derived peptidebound by gp120 of HIV (from the V3 domain of preferably fusin (Feng etal., 1996, Science 272:872-877) or the CC CKR-5 receptor (Samson et al.,1996, Biochemistry 35:3362-3367)). In another specific embodiment, theadministered composition comprises NeuAcα2-6Galβ1-4Glc ligand forinfluenza virus, a recessed hemagglutinin ligand which is conservedamong the main serotypes of the virus.

In another embodiment, an αGal oligosaccharide composition of theinvention comprising a ligand bound by a parasite is administered totarget anti-αGal antibody-mediated lytic attack to the parasite. In aspecific embodiment, the administered composition comprises NeuAcα2-3Galand thereby directs the complement-mediated lytic attack of cellsexpressing the trypanosoma trans-sialidase that binds NeuAcα2-3Gal.

Modifications of the invention in addition to those described above fortreating cancer, autoimmunity disorders, immunosuppression, viraldiseases and parasitic diseases will become apparent to those skilled inthe art form the foregoing description. Additionally, upon reading thepresent disclosure, it will become apparent to those skilled in the artthat the compositions of the invention may be administered to targetanti-αGal antibody-mediated lytic attack to any therapeuticallysignificant target for which a "homing molecule" (e.g., monoclonalantibody, receptor ligand etc.) has been or can be routinely identified,isolated and/or generated. Such modifications are intended to fallwithin the scope of the appended claims.

5.6. THERAPEUTIC COMPOSITIONS AND METHODS OF ADMINISTERING

The pharmaceutical compositions of the invention are useful inattenuating xenograft rejection and/or targeting anti-αGal directedcomplement-mediated lytic attack of targeted tissue and cell types.These compositions contain as an active ingredient, one or more distinctαGal oligosaccharides and/or pharmaceutically acceptable derivativesthereof. The αGal oligosaccharide composition of the invention can beadministered to a patient either by itself, in combination with otherpharmaceutical agents, and/or in pharmaceutical compositions where it ismixed with suitable carriers or excipient(s).

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Effective dosages ofthe αGal compositions of the invention to be administered may bedetermined through procedures well known to those in the art whichaddress such parameters as biological half-life, bioavailability, andtoxicity. Such determination is well within the capability of thoseskilled in the art, especially in light of the detailed disclosureprovided herein. For example, from the data presented in Section 6, itis determined that monovalent αGal pentasaccharide and αGaltrisaccharide are efficacious in vivo at doses required to achievecirculating concentrations of 1 mM or greater. In addition to the activeingredients these pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically.

5.6.1. DOSAGE

According to the method of the invention, attenuation of the hyperacuterejection of a xenograft is achieved by the administration of atherapeutically effective amount of an αGal oligosaccharide and/orpharmaceutically acceptable derivative of the invention, i.e., a dosesufficient to bind to and/or neutralize anti-αGal antibodies in theserum of a patient. For example, monovalent αGal pentasaccharide ormonovalent αGal trisaccharide may be administered as an infusions toattain steady state serum concentrations of 0.5-12 mM for 9-21 days,preferably for at least 14 days. Preferable blood concentrations of themonovalent αGal trisaccharide or αGal pentasaccharide are from 1-1.5 mMor 1.5-2.0 mM. A most pref erred dosage is about 1 mM.¹ Doses formultivalent αGal trisaccharide or αGal pentasaccharide are expected tobe lower (in the μM αGal epitope range) and may be determined usingtechniques known in the art. Desirable blood levels may be maintained bya continuous infusion of the αGal oligosaccharide and/orpharmaceutically acceptable derivative comprising compositions of theinvention as ascertained by plasma levels measured by techniques knownin the art, such as HPLC (see e.g., Fu et al., U.S. application Ser. No.08/563,822, filed Nov. 28, 1995, which is herein incorporated byreference in its entirety). Alternatively, doses of an αGaloligosaccharide and/or pharmaceutically acceptable derivative of theinvention may be administered in intervals of from about once per day to4 times per day. For example, a preferred dose is administered toachieve steady state serum concentrations of monovalent αGaltrisaccharide, αGal pentasaccharide, or a pharmaceutically acceptablederivative thereof, of 1-1.5 mM or 1.5-2.0 mM. A most preferred dosageachieves a steady state serum concentration of about 1 mM. This may beachieved by the sterile injection of a 2.0% solution of the administeredingredients in buffered saline (any suitable saline solutions known tothose skilled in the art of chemistry may be used).

When administering multivalent αGal oligosaccharides, it is preferredthat dosage is carefully titrated upwardly and the serum is monitored tominimize immune complex formation.

Effective amounts of the therapeutic agents, e.g., classicalimmunosuppressive agents to be used in combination with the αGaloligosaccharide compositions of the invention are based on therecommended doses known to those skilled in the art for the such agents.For example, doses for cyclosporine would be directed toward maintaininga whole blood level of 200-300 mg/ml, as measured by HPLC;cyclophosphamide at the dosage of 0.5-2 mg/kg/day; and prednisone at 1mg/kg/day in divided doses (Cooper, D. K. C., Immediate PostoperativeCare and Maintenance Immunosuppressive Therapy, pp. 89-100 in Cooper, D.K. C. and Novitzky, D., eds., The Transplantation and Replacement ofThoracic Organs (Kluwer, Dordrecht 1990)). Minimization of possibleside-effects can be found in standard physician reference texts. Itshould be noted that the attending physician would know how to and whento terminate, interrupt, or adjust therapy to lower dosage due totoxicity, bone marrow, liver or kidney dysfunctions or other adversedrug interaction. Conversely, the attending physician would also know toadjust treatment to higher levels if the clinical response is notadequate (precluding toxicity).

A therapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of signs and symptoms associatedwith xenograft rejection, or a prolongation of xenograft survival in apatient. In applications where pharmaceutical compositions of theinvention target anti-αGal antibody complement-mediated lytic attack oftargeted tissues or cell types, a therapeutically effective dose refersto that amount of the compound sufficient to prevent activation of thecomplement pathway. Toxicity and therapeutic efficacy of such compoundscan be etermined by standard pharmaceutical procedures in cell culturesor experimental animals that produce anti-αGal antibodies, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀ /ED₅₀. Compounds whichexhibit large therapeutic indices are preferred. The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosage for use in humans. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays using cells that express αGal antigenic determinants ontheir surface, such as, for example, porcine PK-15 pig kidneyendothelial cells, pig aortic endothelial cells, and murine MAE cells.Preferably, these cells are from the same species as the donor and mostpreferably from the same tissue-type as the tissue to be transplanted. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves in a half-maximal neutralization and/orbinding of anti-αGal antibodies in serum of a patient compared to acontrol that has not been treated with the αGal oligosaccharidecompositions of the invention, as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. For example, Table 2 in Section 6.6 provides estimated doses ofmonovalent αGal trisaccharide and αGal pentasaccharide required toobtain steady state serum concentrations of 0.5, 1.0 and 2.0 mM. Dosescalculated in Table 2 were based on in vivo serum analysis of thepharmokinetic profile (See Table 1) and serum anti-αGal antibodyneutralizing capacity in baboons of monovalent αGal trisaccharides andαGal pentasaccharides. Levels in plasma may be measured, for example, byhigh performance liquid chromatography (HPLC). See e.g., Fu et al., U.S.application Ser. No. 08/563,822, filed Nov. 28, 1995, which is hereinincorporated by reference in its entirety. The exact formulation, routeof administration and dosage can be chosen by the individual physicianin view of the patient's condition. (See e.g., Fingl et al., 1975, in"The Pharmacological Basis of Therapeutics", Ch. 1 p1).

5.6.2. ROUTES OF ADMINISTRATION

Pharmaceutical compositions comprising αGal oligosaccharide orpharmaceutically acceptable derivative thereof can be administered to apatient, preferably a human or old world monkey, by itself, or inpharmaceutical compositions where it is mixed with suitable carriers orexcipient(s) at doses to ameliorate symptoms associated with xenograftrejection, prolong xenograft survival in a patient or to directcomplement-mediated lytic attack of targeted tissue or cell types.

Use of pharmaceutically acceptable carriers to formulate the compoundsherein disclosed for the practice of the invention into dosages suitablefor systemic administration is within the scope of the invention. Withproper choice of carrier and suitable manufacturing practice, thecompositions of the present invention, in particular, those formulatedas solutions, may be administered parenterally, such as by intravenousinjection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for the form of administration desired.

The pharmaceutical compositions of the invention may be administeredusing techniques well known to those in the art. Preferably agents areformulated and administered systemically. Techniques for formulation andadministration of the compounds of the invention may be found in"Remington's Pharmaceutical Sciences," 18th ed., 1990, Mack PublishingCo., Easton, Pa, latest edition. Suitable routes of administration may,for example, include oral, rectal, transmucosal, or intestinaladministration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections; transdermal, topical, vaginal and the like. Thepreferred routes of administration are by intravenous infusion,intravenous injection, and intramuscular injection. Dosage forms includebut are not limited to tablets, troches, dispersions, suspensions,suppositories, solutions, capsules, gels, syrups, slurries, creams,patches, minipumps and the like.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained in the form of a solid excipient, optionally bygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are, in particular, fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration,the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. The cosolventsystem may be the VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5%dextrose in water solution. This co-solvent system dissolves hydrophobiccompounds well, and itself produces low toxicity upon systemicadministration. Naturally, the proportions of a co-solvent system may bevaried considerably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols. Pharmaceutical compositions suitable foruse in the present invention include compositions wherein the activeingredients are contained in an effective amount to achieve its intendedpurpose. Determination of the effective amounts is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For clarity of discussion, the invention is described in the subsectionsbelow by way of example for the αGal pentasaccharide to neutralizeanti-αGal antibodies and to attenuate xenograft rejection. However, theprinciples disclosed herein may be applied to other αGaloligosaccharides in attenuating xenograft rejection and in the use ofcompositions comprising αGal oligosaccharides to targetcomplement-mediated lytic attack to specific cells containing a surfacemarker, such as, for example, anti-αGal antibody producing B-cells.

6. EXAMPLES

6.1. ENZYMATIC SYNTHESIS OF αGAL TRISACCHARIDE

N-acetylglucosamine (GlcNAc) was dissolved in 5 mM sodium phosphatebuffer at 15 mM, and UDP-Gal, synthesized organically, using techniquesknown in the art, was then added to a final concentration of 5 mM. Abacterial lysate containing recombinant Neisseria polysacchareaβ1,4-galactosyltransferase was then added to this mixture to a finalactivity of 10 μmoles/min/L. The DNA encoding thisβ1,4-galactosyltransferase was generated by the polymerase chainreaction (PCR) using techniques known in the art (see e.g., Innis, M.,1990, PCR protocols, a Guide to Methods and Applications, AcademicPress, California; and Dieffenbach, W., 1995, PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, New York). The PCR reaction usedprimers corresponding to sequences flanking theβ1,4-galactosyltransferase of Neisseria gonorrhoea (see U.S. Pat. No.5,545,553) and Neisseria polysaccharea template DNA that was isolatedusing standard methods known in the art (see e.g., Maniatis, T., 1982,Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.; Current Protocols in MolecularBiology). The PCR generated DNA was then cloned into the pGEX expressionvector (Pharmacia) using methods known in the art (see e.g., Maniatis,T., 1982, Molecular Cloning, A Laboratory Manual, 2d ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology). The protein expressed by this expression constructis a fusion protein in which glutathione-S-transferase is fused to theamino terminal end of the β1,4-galactosyltransferase. Lysates containingthis fusion protein were prepared from E. coli host cells that weretransformed with the expression construct and in which expression of theconstruct had been induced through the addition ofisopropylthiogalactoside (IPTG).

After 24 h at ambient temperature the reaction mixture containingN-acetyl-lactosamine (LacNAc) and unreacted starting materials wasfiltered to remove proteins and purified by cation (Dowex 50) followedby anion (Dowex 1) exchange chromatography with water elution. Theflow-through material containing LacNAc was pooled, concentrated anddiafiltered by reverse osmosis (RO).

Concentrated aqueous LacNAc was dissolved in phosphate buffer to a finalconcentration of 5 mM, and UDP-Gal, synthesized organically, usingtechniques known in the art, was then added to a final concentration of6 mM. A bacterial lysate containing recombinant mouseα1,3-galactosyltransferase was then added to this mixture to a finalactivity of 15 μmoles/min/L. The DNA encoding thisα1,3-galactosyltransferase was generated by the reverse-transcriptionpolymerase chain reaction (RT-PCR) using techniques known in the art(see e.g., Innis, M., 1990, PCR protocols, a Guide to Methods andApplications, Academic Press, California; and Dieffenbach, W., 1995, PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, New York). Thenucleic acid sequence encoding the mouse α1,3-galactosyltransferase genehas previously been reported (Larsen, et al., 1989 PNAS 86:8227-8231).Primers used in the RT-PCR reaction were designed so as to amplify acDNA fragment corresponding to the mouse α1,3-galactosyltransferasegene, but which lacks nucleic acid sequences encoding the first 60 aminoacids of the protein, i.e., nucleic acids encoding the amino terminalcytoplasmic tail and membrane spanning region of the enzyme. TemplatemRNA used in the RT-PCR reaction was isolated from mouse kidney usingstandard methods known in the art (see e.g., Maniatis, T., 1982,Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.; Current Protocols in MolecularBiology). The PCR generated cDNA was then cloned into the pGEXexpression vector (Pharmacia) using methods known in the art (see e.g.,Maniatis, T., 1982, Molecular Cloning, A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology). The protein expressed by this expression constructis a fusion protein in which glutathione-S-transferase is fused to theamino terminal end of the modified α1,3-galactosyltransferase encoded bythe PCR generated cDNA. Lysates containing this fusion protein wereprepared from E. coli host cells that were transformed with theexpression construct and in which expression of the construct had beeninduced through the addition of IPTG.

After 24 h at 37° C., the reaction mixture, containing αGal-LacNAc andunreacted starting materials was filtered and purified by cation (Dowex50) followed by anion (Dowex 1) exchange chromatography with waterelution. The flow-through material was concentrated by RO. Theconcentrated α-Gal-LacNAc solution was mixed with acetonitrile (30:70),applied to a preparative amino-bonded silica HPLC column, and elutedisocratically. The fractions containing α-Gal-LacNac (αGaltrisaccharide) were pooled, concentrated and lyophilized.

6.2. ENZYMATIC SYNTHESIS OF αGAL PENTASACCHARIDE

Lactose was dissolved in 5 mM sodium phosphate buffer at mM, andUDP-GlcNAc, obtained by fermentation, using techniques known in the art,was then added to a final concentration of 5 mM. A bacterial lysatecontaining recombinant Neisseria polysacchareaβ1,3-N-acetylglucosaminyltransferase was then added to this mixture to afinal activity of 10 μmoles/min/L. The DNA encoding thisβ1,3-N-acetylglucosaminyltransferase was generated by the polymerasechain reaction (PCR) using techniques known in the art, with primerscorresponding to sequences flanking theβ1,3--N-acetylglucosaminyltransferase of Neisseria gonorrhoea (see U.S.Pat. No. 5,545,553) and Neisseria polysaccharea template DNA that wasisolated using standard methods known in the art. The PCR generated DNAwas then cloned into the pGEX expression vector (Pharmacia) usingmethods known in the art. The protein expressed by this expressionconstruct is a fusion protein in which glutathione-S-transferase isfused to the amino terminal end of theβ1,3-N-acetylglucosaminyltransferase. Lysates containing this fusionprotein were prepared from E. coil host cells that were transformed withthe expression construct and in which expression of the construct hadbeen induced through the addition of IPTG.

At 24 h, β-galactosidase was added to break down unreacted lactose.After 48 h at ambient temperature, the reaction mixture containingLNT-II, the product of the reaction, and unreacted starting materialswas filtered to remove proteins and purified by cation (Dowex 50)followed by anion (Dowex 1) exchange chromatography with water elution.The flow-through material containing LNT-II was pooled, concentrated,and diafiltered by RO.

Concentrated aqueous LNT-II was dissolved in phosphate buffer to a finalconcentration of 5 mM. UDP-Gal, synthesized organically, usingtechniques known in the art, was added to a final concentration of 6 mM.Recombinant Neisseria polysaccharea β1,4-galactosyltransferase,generated as described in Section 6.1, was then added as bacteriallysate to a final activity of 10 μmoles/min/L. After 24 h at ambienttemperature, the reaction mixture, containing LNnT and unreactedstarting materials was filtered to remove proteins, and purified bycation (Dowex 50) followed by anion (Dowex 1) exchange chromatographywith water elution. The flow-through material containing LNnT wasconcentrated by RO.

Concentrated aqueous LNnT was dissolved in phosphate buffer to a finalconcentration of 5 mM, and UDP-Gal, synthesized organically, usingtechniques known in the art, was then added to a final concentration of6 mM. A bacterial lysate containing recombinant mouseα1,3-galactosyltransferase, generated as described in Section 6.1, wasthen added to this mixture to a final activity of 15 μmoles/min/L.

After 24 h at 37° C. the reaction mixture, containing αGal-LNnT andunreacted starting materials, was filtered to remove proteins, andpurified by ion exchange chromatography with water elution. Theflow-through material was concentrated by RO. The concentratedα-Gal-LNnT solution was mixed with acetonitrile (30:70), applied to apreparative amino-bonded silica HPLC column, and eluted isocratically.The fractions containing αGal-LNnT (αGal pentasaccharide) were pooled,concentrated and lyophilized.

6.3. IN VITRO LAMININ ELISA ANALYSIS

In vitro laminin ELISA evaluation of αGal oligosaccharides asneutralizers of anti-αGal antibodies. Mouse laminin is a basementmembrane glycoprotein that expresses 50-70 oligosaccharides terminatingin Galα1-3Gal, i.e., the αGal motif (Arumugham et al., 1986, Biochem.Biophys. Aeta 883:112). In this assay, immobilization of mouse lamininon microtiter plates serves as a capture ligand for anti-αGalantibodies. This example shows that the binding of anti-αGal antibodiesis inhibited to different extent by αGal oligosaccharides of differentlengths and glycosidic bond structure and that the non-αGaloligosaccharides tested do not inhibit antibody binding. The exampleprovides a method by which to determine the ability of αGaloligosaccharides and pharmaceutically acceptable derivative of theinvention to bind to anti-αGal antibodies and to thereby remove theseantibodies from the serum, neutralize anti-αGal antibodies, and/ortarget anti-αGal directed complement-mediated lytic attack to specifictissue or cell types. The example also provides a method by which theeffectiveness of the methods of the invention can be evaluated anddosages determined.

6.3.1. MATERIALS AND METHODS

Sera were mixed with serial dilutions of αGal oligosaccharides(αGal-CHO) at 37° C. for 1 hour, then added to mouse laminin-coatedmicrotiter plates and incubated for 1 hour to allow binding. Themicrotiter plates were coated with 20 μg/mL mouse laminin in 50 mMNaHCO₃ buffer, pH 9.5 at 4° C. overnight, blocked with SuperBlocksolution (Pierce) for 2 hours, and then washed 3 times with SuperBlocksolution. After washing excess serum away, bound IgG and IgM wasmeasured by adding goat anti-human IgG (or IgM) conjugated to alkalinephosphatase and incubating for 1 hour, washing, and adding substrate(pNPP).

6.3.2. RESULTS

Binding of anti-αGal antibodies is inhibited to different extent by αGaloligosaccharides of different lengths (FIG. 1A and FIG. 1B). Non-αGaloligosaccharides (e.g., sucrose (represented by open squares) andGalβ1-4Gal, lactose, and Galβ1-4GlcNAcβ1-3 Galβ1-4Glc (data not shown)do not inhibit antibody binding.

Generally, the αGal trisaccharide (Galα1-3Galβ1-4GlcNAc, represented bythe open circle) is a slightly better inhibitor of IgG (FIG. 1A) and IgM(FIG. 1B) binding to laminin than αGalpentasaccharide(Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc, represented by theopen triangle), which is a slightly better inhibitor of both IgG and IgMbinding to laminin than αGal disaccharide (Galα1-3Gal, represented bythe open diamond).

6.4. IN VITRO IMMOBILIZED αGAL OLIGOSACCHARIDE ELISA ANALYSIS

In vitro immobilized αGal oligosaccharide ELISA analysis evaluation ofanti-αGal antibody titer and of αGal oligosaccharides as neutralizers ofanti-αGal antibodies. This example provides an assay that may be appliedto determine the ability of αGal oligosaccharides and pharmaceuticallyacceptable derivative of the invention to bind to anti-αGal antibodiesand to thereby remove these antibodies from the serum or plasma,neutralize anti-αGal antibodies, and/or target complement-mediated lyticattack to specific tissue or cell types. Such an ELISA is additionallyuseful in clinical monitoring of anti-αGal antibodies in blood ofpatients in advance of, and following xenotransplantation of organs fromanimals that express the αGal epitope.

6.4.1. MATERIALS AND METHODS

The derivative of the αGal pentasaccharide (αGal pentasaccharideglycinamide) was synthesized and immobilized on N-oxysuccinimidemicrotiter plates (Costar) (0.5 mg/ml αGal pentasaccharide glycinamide)in bicarbonate buffer, pH 9.0, overnight at 4° C.; the plate was blockedfirst for 1 hour with 1M ethanolamine in the same buffer and secondlywith phosphate buffered saline, pH 7.4, containing 1% bovine serumalbumin and 0.05% Tween 20 and 0.02% NaN₃ or PBNT. PBNT was used for allthree subsequent plate washes and as a diluent for the antibodies. Serumfrom a baboon was added for 1 hour following pre-incubation with variousoligosaccharides for 1 hour at 37° C., and subsequently washed threetimes. Bound antibodies were identified by the addition of a goatanti-human IgG and IgM-alkaline phosphatase and incubated at roomtemperature for 1 hour, followed by the addition of the substrate pNPP.The optical density at 405 nM was recorded.

6.4.2. RESULTS

Serum IgG and IgM binding to immobilized αGal pentasaccharideglycinamide was not inhibited by sucrose, and minimally by Galβ1-4Gal(FIG. 2A and 2B). All αGal oligosaccharides tested inhibited binding ofIgG and IgM. IgG binding was inhibitable to a greater extent than IgM bymost αGal oligosaccharides, and its binding was also more sensitive todifferences in the structure of the αGal oligosaccharides tested.

6.5. IN VITRO CYTOTOXICITY ANALYSIS

In vitro cytotoxicity evaluation of αGal oligosaccharides asneutralizers of anti-αGal antibodies. This example shows thatcytotoxicity is inhibited slightly better by αGal trisaccharide than byαGal pentasaccharide.

6.5.1. MATERIALS AND METHODS

Sera were incubated with serial dilutions of αGal oligosaccharides at37° C. for 1 hour, then added to αGal-expressing monolayers (pig kidneycells, PK-15) in Terasaki plates for 1 hour at 37° C. Cytotoxicity wasmediated by either endogenous complement, or the sera werede-complemented (heat inactivated for 30 minutes at 56° C.) andexogenous (rabbit) complement was added. After washing away excessserum, the viable dye mix "live-dead" (calcein AM/ethidium homodimer(Molecular Probes Inc., Eugene Oreg.)) was added and monolayers werescored for viability by fluorescence microscopy.

6.5.2. RESULTS

The cytotoxicity results observed in representative individual humans ispresented in FIG. 3A and FIG. 3B. Generally, the αGal trisaccharide(Galα1-3Galβ1-4GlcNAc) is a slightly better inhibitor of cytotoxicitythan αGal pentasaccharide (Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc). Similarresults were obtained using mouse endothelial cells (MAE) or primary pigaortic endothelial cell cultures (data not shown).

6.6. IN VIVO BLOOD SERUM ANALYSIS

Pharmokinetics of αGal trisaccharides and αGal pentasaccharides inbaboons. This example presents a study of the pharmokinetic profile andserum anti-αGal antibody neutralizing capacity in baboons of αGaltrisaccharides and αGal pentasaccharides and previews the compounds'suitability for use in the prevention of hyperacute rejection of porcineorgans xenotransplanted into baboons. The study reveals that thepharmokinetics of the αGal trisaccharides and αGal pentasaccharides inbaboons are similar and that a minimal serum concentration of 1 mM ofthe αGal trisaccharide or αGal pentasaccharide is required to inhibithyperacute rejection resulting from anti-αGal antibody directedcomplement-mediated lytic attack.

6.6.1. MATERIALS AND METHODS

The pharmacokinetics of the αGal trisaccharide compositions and the αGalpentasaccharide compositions were determined by delivering to one baboonby 15 minute intravenous bolus, 0.5 mMol/Kg of one oligosaccharide and48 hours later, delivering 0.5 mmol/Kg of the second oligosaccharide,and for a second baboon, delivering the oligosaccharides according tothe same procedure, but in the reverse order.

Three Baboons were outfitted with indwelling venous and arterialcatheters to the femoral vessels, and held in place with the aid of ajacket. Oligosaccharides were administered through the catheter in a 15minute continuous infusion. Blood samples (as plasma) were collected forthe determination of oligosaccharide concentration by HPLC and (asserum) for the determination of antibody titers by ELISA andcytotoxicity (using techniques described in Sections 6.3, 6.4 and 6.5),as well as for complement and blood chemistry, at predetermined timeintervals and volumes.

6.6.2. RESULTS

The analysis of reactive anti-αGal antibody by ELISA (binding to mouselaminin, αGal albumin neoglycoconjugates and PK-cells) indicate thatwith enough added αGal antibody oligosaccharide, αGal antibody bindingand cytotoxicity can be inhibited (See e.g., FIG. 4, which showsinhibition of anti-αGal antibody cytotoxicity of MAE cells atconcentrations of 1.0-2.0 mM in one of the baboons tested. The volume ofdistribution governs the magnitude of the plasma concentration at time 0after bolus drug administration. It is used to calculate a loading dose.During a continuous infusion, the drug's clearance governs thesteady-state concentration for a given infusion rate. The clearance isused to calculate the infusion rate needed to maintain a desired drugconcentration. The half-life describes how quickly concentrationsdiminish with time. Mathematical formulations known in the art wereapplied to estimate the pharmacokinetic parameters of the αGaltrisaccharide and αGal pentasaccharide. The pharmacokinetic parameterdata, presented in Table 1, indicates that the pharmacokinetics of theαGal trisaccharide and αGal pentasaccharide in baboons are similar.Compared to other drugs, the volumes of distribution of theseoligosaccharides are low, less than the extravascular water volume (<400mL/kg). The clearance is typical of compounds eliminated by GFR(glomerular filtration rate). The half-life of these drugs would beexpected to be about 50% longer in humans due to a lower rate ofclearance.

                  TABLE I                                                         ______________________________________                                        Pharmacokinetic parameter estimates                                           Pharmacokinetic       αGal-LNnT                                                                          αGal-LacNAc                            Parameter  Units      (1003, 1015)                                                                             (1003, 1015)                                 ______________________________________                                        Volume of  mL/kg      199, 230   288, 362                                     Distribution                                                                  Clearance  mL/min/kg  2.93, 3.97 4.18, 4.88                                   Half-Life  min        47.3, 40.2 46.8, 51.5                                   ______________________________________                                    

The pharmacokinetic data presented in Table 1 were used to predictconcentrations of monovalent αGal trisaccharide and αGal pentasacchariderequired to attain steady state serum concentrations of 0.5, 1.0 and 2.0mM, respectively, by mathematical formulations known in the art. Thisdata is presented in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Calculated αGal trisaccharide and αGal                            pentasaccharide administration to attain 0.5, 1.0,                            and 2.0 mM steady state serum concentrations                                                αGal-trisaccharide                                                                  αGal pentasaccharide                          __________________________________________________________________________    Desired steady-state                                                                        0.5 1.0 2.0 0.5 1.0 2.0                                         serum concentration (mM)                                                      Desired steady-state                                                                        0.26                                                                              .52 1.05                                                                              0.45                                                                              0.9 1.79                                        serum concentration (mg/mL)                                                   LOADING DOSE  191 382 764 206 412 824                                         (bolus or 15 min                                                              infusion); mg/Kg                                                              g/20 Kg baboon                                                                              3.82                                                                              7.64                                                                              15.28                                                                             4.12                                                                              8.24                                                                              16.48                                       CONTINUOUS INFUSION                                                                         2.57                                                                              5.14                                                                              10.28                                                                             3.56                                                                              7.12                                                                              14.24                                       mg/min/Kg                                                                     mg/h/Kg       154.2                                                                             308.4                                                                             616.8                                                                             213.6                                                                             427.2                                                                             854.4                                       g/h/20 Kg     3.08                                                                              6.17                                                                              12.34                                                                             4.27                                                                              8.54                                                                              17.09                                       g/24 h/20 Kg  74.0                                                                              148 296.1                                                                             102.6                                                                             205.2                                                                             410.1                                       Kg/14 days    1.04                                                                              2.08                                                                              4.15                                                                              1.44                                                                              2.88                                                                              5.74                                        __________________________________________________________________________

6.7. IN VIVO XENOTRANSPLANTATION

Primate model for testing and application of αGal oligosaccharides anddosages thereof. Due to the many similarities between human and baboonimmune systems (Neubauer et al., 1981, J. Immunogenetics, 8:433-442;Garver et al., 1980, Cytogenetics & Cell Genetics, 27:238-245; Brodskyet al., 1982, Immunogenetics, 155:151-166; Hammer, C., in Hardy, M. A.(ed.), Xenograft 25, 115-123 (Elsevier, N.Y., 1989); Stark, J. H., etal., Transplantation, 30 52(6):1072-1078 (December 1991); Hammer, C., inCooper, D. K. C., et al. (eds.), Xenotransplantation, 429-438(Springer-Verlag 1991)), and because of the large size of baboons, theseanimals are convenient experimental model recipients of pig organs.These non-human primates also express anti-pig antibodies, and rejectpig organs hyperacutely (Lexer et al., 1986, J. Heart Transplant,4:411-418; Ye, Y., Cooper, D. K. C., in Cooper, D. K. C., et al. (eds.),Xenotransplantation, 389-393 (Springer-Verlag 1991); Cooper et al.,1991, J. Heart Transplant, 7:238-246, 1988; Platt et al., 1991,Transplantation, 52(2):214-220). A normal porcine heart was transplantedinto the neck of a normal baboon. αGal pentasaccharide was infused(i.v.) to establish and maintain blood oligosaccharide levels predictedto inhibit HAR until infusate was exhausted (See Table 2). The abilityof i.v. infused αGal pentasaccharide to block hyperacute rejection (HAR)of porcine heart xenografted to the neck of a baboon was determined andblood levels of αGal oligosaccharide, anti-αGal antibodies, and serum orplasma cytotoxicity toward pig kidney and mouse endothelial cells wasmonitored and correlated with the onset of HAR.

6.7.1. MATERIALS LAND METHODS

XENOGRAFT HEART TRANSPLANTATION

The heart was excised from the pig donor and transplanted in the neck ofthe baboon recipient using techniques essentially as described by Cooperet al., 1993, Transplantation, 56:769-777.

OLIGOSACCHARIDE ADMINISTRATION

The two xenotransplant baboons were outfitted with an indwellingcatheter to the femoral vein to infuse compound and a catheter to thefemoral artery for blood sampling. Following the transplant procedure,but just prior to providing circulatory access to the xenograft, theoligosaccharide was administered by i.v. pump through the catheter in a15 min loading dose, followed by continuous 4 hour infusion to achieve2.5 mM blood concentration (as determined by calculations presented inTable 2). Blood samples (as plasma) were collected for the determinationof oligosaccharide concentration by HPLC and (as serum) for thedetermination of antibody titers by ELISA and cytotoxicity (usingtechniques described in Sections 6.3, 6.4 and 6.5 infra), as well as forcomplement and blood chemistry, at predetermined time intervals. Bloodspecimens collected for plasma were immediately centrifuged and theplasma withdrawn and stored frozen at -20° C. Blood specimens collectedfor serum were clotted at 4° C. overnight and the withdrawn serum frozenat -20° C. The αGal pentasaccharide was prepared according to theenzymatic methods set forth in example 6.2 and stored as a dry, white,solid powder. The oligosaccharide was dissolved in 250 mL sterileinfusion grade saline to a concentration of 0.154 g/mL, or 177 mM.Induction anesthesia was with ketamine hydrochloride 5 mg/kg/body weightin and 0.5 mg/kg of xylazine iv. Intravenous fluid of approximately 20ml/kg body weight was given during the course of the operative procedurethrough either a peripheral or central vein. Atropine 0.5 mg/kg wasgiven iv. The larynx was sprayed with 2% lidocaine. Endotrachealintubation was carried out. The endotracheal tube was taped to themaxilla. The pig or baboon was ventilated with a Harvard positivepressure ventilator. Anesthesia was maintained with nitrous oxide 2L/min and oxygen 1 L/min and 0.2%-1.2% halothane, at a respiratory rateof approximately 10 to 20 breaths per minute depending on the size ofthe animal. Tidal volume (approximately 20-24 ml/kg) was also adjustedto suit the size of the animal, and the ventilatory pressure wasadjusted to approximately 15 to cm H₂ O. Blood gases were checked, andventilation adjusted as necessary. Arterial pressure was monitored inthe recipient baboon by intermittent automatic cuff recordings.

The intra-arterial line in the femoral artery allowed continuousmonitoring of arterial pressure, and access for determination of bloodgases. A suitable antibiotic was administered i.v. to the recipientbefore any incision was made, and was then administered i.v. at 12hourly intervals for 72 hours. Blood cultures were taken on the dayfollowing the operation to ensure that further antibiotic therapy wasnot indicated.

After αGal pentasaccharide infusion, when the xenograft's heartbeatbecame weak and irregular, external color became blotchy, and swellingof the atria was observed, the donor heart was removed under ketaminesedation after removing the skin sutures to expose the heart and placingligatures around the common carotid artery and internal jugular veinabove and below the sites of anastomosis. The ligatures were tied down,and the heart excised.

The heart was divided at midventricular level, and clot and blood washedout with saline. A thin section across the ventricles was taken and sentfor histopathological examination to confirm the presence or otherwiseof rejection.

6.7.2. RESULTS

The only significant alteration in blood chemistry that was recorded wasan increase in creatinine phosphokinase, which is a natural consequenceof surgery. Hematology results were unremarkable. The pig hearts of thetwo baboons resumed beating shortly after the baboon blood flow waschanneled through the xenograft. One heart had to be electricallyshocked to initiate regular beating. Once the heartbeat resumed, regularcolor returned to the heart and an even beat was maintained for theduration of the four hour oligosaccharide infusion. At the end ofinfusion, heart beat became weak and irregular, external color becameblotchy, and swelling of the atria was observed. At this time, more than1 hour following cessation of the 4 hour αGal pentasaccharide infusion,the xenograft was excised. Hearts from normal pigs xenotransplanted intobaboons in this fashion in the past, but without interference withimmune functions, have been hyperacutely rejected without exception in5-10 minutes. Analysis of reactive anti-αGal antibody by ELISA (bindingto mouse laminin, α-Gal-albumin neoglycoconjugates and PK-15 cells)indicated pronounced reduction in the anti-αGal antibody titercoinciding with the period during which serum concentration of αGaloligosaccharide exceeded 1 mM (Data not shown).

Cytotoxicity of the baboon sera against MAE cells was reduced by 100%and against PK-15 by 85-95% during the 4 hours of αGal-LNnT infusion(FIGS. 5A and 5B). HPLC analysis of plasma samples revealed higher thanexpected blood levels of oligosaccharide in both xenotransplantedbaboons. Blood concentrations increased steadily following release ofblood flow through the pig heart reaching a plateau of 12 mM in thefirst baboon (FIG. 5A) and 5 mM in the second (FIG. 5B). These bloodconcentrations were approximately twice what was predicted by theearlier pharmacokinetic infusions in the same baboons in the absence ofxenografting or anesthesia. It is probable that increased blood flowthrough the xenograft, which acts as an arterial-venous shunt, as wellas the peripheral vasodilatation caused by the anesthetic isoflurene,resulted in a reduction in renal filtration rate. This effect wouldresult in the reduced clearance rate of the infused oligosaccharide, andgive rise to the observed accumulation of oligosaccharide in thebaboons' circulation.

Essentially normal histology was observed during the initial 3 hours ofinfusion (data not shown). Only when the xenografts exhibited externaldeterioration was there evidence of vascular congestion, vascular andintersticial neutrophilia, and, in one baboon, fibrin deposition at theluminal surface of the endothelium. Immunofluorescence indicated IgG,IgM and complement (C3) deposition at the endothelial surface as earlyas 1.5 hours in the first baboon and not until 3 hours in the secondbaboon.

6.8. EXTRACORPOREAL DEPLETION OF ANTI-αGAL ANTIBODIES

Ex vivo depletion of anti-αGal antibodies from human serum by passageover αGal sepharose. This example shows that most of the cytotoxicantibody population in human serum can be absorbed upon perfusionthrough columns of a matrix containing αGal trisaccharide or αGalpentasaccharide.

6.8.1. MATERIALS AND METHODS

Pooled human serum was diluted 1:1 with phosphate buffered saline andpassed over minicolumns (0.3 mL bed) containing beads to which αGalLacNAc (αGal trisaccharide), αGal-LNnT (αGal pentasaccharide) or glucosewas coupled. Coupling of the αGal oligosaccharide compositions to thesepharose matrix was accomplished by reacting glycine amides of thecarbohydrates with sepharose-bearing N-hydroxysuccinimide groups (Sigma)at pH 9.7 in sodium borate buffer, overnight at 4° C. Sepharose beadsbearing glucose were purchased from Sigma.

Fractions were tested for cytotoxicity against pig PK-15 cells bylive/dead assays as described in Sections 6.3, 6.4 and 6.5.

6.8.2. RESULTS

The cytotoxicity data observed for two different lots of serum passagedover beads bearing immobilized αGal-LacNAc and αGal-LNnT, is presentedin FIG. 6A and FIG. 6B. The early fractions from the αGaloligosaccharide-immobilized beads exhibited greatly diminishedcytotoxicity, indicating that the anti-αGal cytotoxic antibodies hadbeen absorbed onto the αGal derivatized beads. The beads covered withαGal trisaccharide (αGal-LacNAc) removed most but not all of thecytotoxic activity from the first 50 mL serum, whereas the beadsderivatized with αGal pentasaccharide (αGal-LNnT) removed nearly all ofthe cytotoxicity in the early fractions, and continued to removecytotoxic antibodies for longer than the αGal trisaccharide derivatizedbeads. As demonstrated in FIG. 6B, beads derivatized with glucose werecomparatively inefficient in removing cytotoxic antibodies.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying figures. Such modificationsare intended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A method for suppressing B-lymphocytes expressinganti-αGal idiotypes in a subject, comprising administering an amount ofa composition comprising an αGal oligosaccharide linked to a cytocidalagent effective in binding anti-αGal idiotypes expressed on the surfaceof B-lymphocytes.
 2. The method of claim 1, further comprising the stepof contacting serum of the subject with an immobilized αGaloligosaccharide ex vivo.
 3. The method of claim 1, wherein the cytocidalagent is selected from the group consisting of ricin A, Pseudomonasexotoxin, cytosine arabinoside and daunorubicin.
 4. A method fortreatment of a parasitic disease in a subject comprising administeringan amount of a composition comprising an αGal oligosaccharide linked toa ligand that binds to a target located on a parasite.
 5. The method ofclaim 4, wherein the target is selected from the group consisting ofNeuAcα2-3Gal, trans-sialidase and a Gal/GalNAc-terminatingoligosaccharide.
 6. The method of claim 4, wherein the parasite isselected from the group consisting of Trypanosoma cruzi, Plasmodiumfalciparum and Entamoeba histolytica.
 7. The method of claim 4, whereinthe parasitic disease is selected from the group consisting of Chagasdisease, malaria and amoebic dysentery.
 8. The method of claim 1 or 4,wherein the αGal oligosaccharide is selected from the group consistingof Galα1-3Gal, Galα1-3Galβ1-4Glc, Galα1-3Galβ1-4GlcNAc,Galα1-3Galβ1-4GlcNAcβ1-3Gal and Galα1-3Galβ1-4GalNAcβ1-3Galβ1-4Glc.